Uses of Ku70

ABSTRACT

This invention provides a method of diagnosing a predisposition to cancer in a subject comprising: (a) obtaining a nucleic acid sample from the subject; and; (b) determining whether one or more of the subject&#39;s Ku70 alleles or regulatory regions to those alleles are deleted or different from the wild type so as to reduce or eliminate the subject&#39;s expression of polypeptide having tumor suppressor activity. This invention also provides a method of assessing the severity of cancer in a subject comprising: (a) obtaining a nucleic acid sample from the subject; and (b) determining whether one or more of the subject&#39;s Ku70 alleles or regulatory regions to those alleles are deleted or different from the wild type so as to reduce or eliminate the subject&#39;s expression of polypeptide having tumor suppressor activity. This invention also provides a method of assessing the severity of cancer in a subject comprising: determining the subcellular localization of Ku70 in the subject, wherein an abnormal subcellular localization of Ku70 indicates a predisposition to cancer.

[0001] This application claims the benefit of U.S. provisionalApplication No. 60/091,188, filed Jun. 30, 1998. The contents of thepreceding application are hereby incorporated into this application byreference.

[0002] The invention disclosed herein was made with Government supportunder NIH Grant Nos. CA-31397 and CA-56909 from the Department of Healthand Human Services and the Department of Energy OHER (DC). Accordingly,the U.S. Government has certain rights in this invention.

[0003] Within this application, publications are referenced withinparentheses. Full citations for these references may be found at the endof each series of experiments. The disclosures of these publications intheir entireties are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art towhich this invention pertains.

BACKGROUND OF THE INVENTION

[0004] Two distinct processes involving DNA double-strand breaks (DSB)have been identified in mammalian cells: the repair of DNA damageinduced by ionizing radiation and V(D)J recombination during T- andB-cell development. So far, all mammalian cell mutants defective in DNADSB repair share the common phenotype of hypersensitivity to radiation,and impaired ability to undergo V(D)J recombination (1-6). Cell fusionstudies using DSB repair mutants of human-rodent somatic hybrids havedefined four complementation groups: IR4, IR5, IR6, and IR7. Genetic andbiochemical analyses have revealed that cells of IRS (e.g., xrs-6) andIR7 (e.g., scid) are defective in components of the DNA-dependentprotein kinase (DNA-PK) (2, 7-9). DNA-PK is a serine/threonine kinasecomprised of a large catalytic subunit (DNA-PK_(cs)) and a DNA-targetingcomponent termed Ku, which itself is a heterodimer of a 70-kDa (Ku70)and a 86-kDa (Ku80) polypeptide (10-12). Recently, DNA-PK_(cs) has beenshown to be the gene responsible for the murine scid (severe combinedimmunodeficiency) defect (13-15); and Ku80 has been identified to beXRCC5 (16-18), the X-ray-repair cross-complementing gene for IR5. Ku80knockout mice were found to exhibit severe combined immunodeficiency,defective processing of V(D)J recombination intermediates, and growthretardation (19, 20).

[0005] Though Ku70 has been designated as XRCC6 (7, 8) and is animportant component of the DNA-PK complex, the function of Ku70 in vivois hitherto unknown. To define the role of Ku70 in DNA repair and V(D)Jrecombination, we targeted the Ku70 gene in mice. Ku70 homozygotesexhibit proportional dwarfism, a phenotype of Ku80^(−/−), but not ofscid mice. Absence of Ku70 confers hypersensitivity to ionizingradiation and deficiency in DNA DSB repair, which are characteristics ofboth Ku80^(−/−) and scid mice. Surprisingly, in contrast to Ku80^(−/−)and scid mice, in which both T- and B-lymphocyte development arearrested at early stage, lack of Ku70 is compatible with T cell receptorgene recombination and the development of mature CD4⁺CD8⁻ and CD4⁻CD8⁺ Tcells. Our data, for the first time, provide direct evidence supportingthat Ku70 plays an essential role in DNA DSB repair, but is not requiredfor TCR gene recombination. These results suggest that distinct butoverlapping repair pathways may mediate DSB repair and V(D)J rejoining;furthermore, it suggests the presence of a Ku70-independent rescuepathway in TCR V(D)J recombination. The distinct phenotype of Ku70^(−/−)mice should make them valuable tools for unraveling the mechanism(s) ofDNA repair and recombination.

[0006] Ku is a complex of two proteins, Ku70 and Ku80, that functions asa heterodimer to bind DNA double-strand breaks (DSB) and activateDNA-dependent protein kinase (DNA-PK). The role of the Ku70 subunit inDNA DSB repair, hypersensitivity to ionizing radiation and V(D)Jrecombination was examined in mice that lack Ku70 (Ku70^(−/−) LikeKu80^(−/−) mice, Ku70^(−/−) mice showed a profound deficiency in DNA DSBrepair and were proportional dwarfs. Surprisingly, in contrast toKu80^(−/−) mice, in which both T- and B-lymphocyte development werearrested at early stage, lack of Ku70 was compatible with T cellreceptor gene recombination and the development of mature CD4⁺CD8⁻ andCD4⁻CD8⁺ T cells. Our data shows, for the first time, that Ku70 plays anessential role in DNA DSB repair, but is not required for TCR V(D)Jrecombination. These results suggest that distinct but overlappingrepair pathways may mediate DNA DSB repair and V(D)J recombination.

SUMMARY OF THE INVENTION

[0007] This invention provides a method of diagnosing a predispositionto cancer in a subject comprising: (a) obtaining a nucleic acid samplefrom the subject; and; (b) determining whether one or more of thesubject's Ku70 alleles or regulatory regions to those alleles aredeleted or different from the wild type so as to reduce or eliminate thesubject's expression of polypeptide having tumor suppressor activity.

[0008] This invention also provides a method of assessing the severityof cancer in a subject comprising: (a) obtaining a nucleic acid samplefrom the subject; and (b) determining whether one or more of thesubject's Ku70 alleles or regulatory regions to those alleles aredeleted or different from the wild type so as to reduce or eliminate thesubject's expression of polypeptide having tumor suppressor activity.

[0009] This invention also provides a method of assessing the severityof cancer in a subject comprising: determining the subcellularlocalization of Ku70 in the subject, wherein an abnormal subcellularlocalization of Ku70 indicates a predisposition to cancer.

[0010] In addition, this invention provides the above-described methods,wherein the abnormal subcellular localization of Ku70 comprisesincreased cytosolic localization of Ku70.

[0011] In addition, this invention provides a method of inhibiting thegrowth of cancer cells, comprising introducing into a cell a Ku70 geneunder conditions permitting expression of the gene.

[0012] This invention also provides the above-described methods, whereinthe cancer is T-cell lymphoma.

[0013] In addition, this invention provides the above-described methods,wherein the cell prior to the introduction of the Ku70 gene wascharacterized as having a mutation at one or more Ku70 alleles orregulatory regions thereto.

[0014] In addition, this invention provides the above-described methods,wherein the cell prior to the introduction of the Ku70 gene wascharacterized as having reduced expression of Ku70.

[0015] This invention further provides the above-described methods,wherein the Ku70 gene is incorporated into an expression vector prior tointroduction into the cell.

[0016] This invention also provides a method of inhibiting the growth ofcancer cells, comprising introducing Ku70 into a cell.

[0017] In addition, this invention provides the above-described methods,wherein the cancer is T-cell lymphoma.

[0018] In addition, this invention provides a transgenic cell, whereinthe expression of the Ku70 allele have been altered to increase thesusceptibility of the cell to DNA damage.

[0019] This invention also provides a transgenic cell, wherein theexpression of the Ku70 allele has been altered to increase thesusceptibility of the cell to cancerous growth.

[0020] This invention also provides a transgenic organism, comprising anorganism whose germ line cells has been altered at the Ku70 allele toproduce an organism whose offspring have an increased likelihood ofdeveloping tumors.

[0021] In addition, this invention provides a transgenic organism,comprising an organism whose germ line cells has been altered at theKu70 allele to produce an organism whose offspring have an increasedlikelihood of having increased susceptibility to DNA damage.

[0022] This invention further provides a method of screening a compoundfor carcinogenic activity, comprising: (a) contacting cells havingreduced expression of Ku70 with the compound; and (b) determiningwhether the compound results in a malignant transformation phenotype.

[0023] This invention also provides a method of screening a compound forability to restore Ku70 activity to cells having Ku70 defect symptomsresulting from reduced Ku70 activity, comprising: (a) contacting cellshaving reduced expression of Ku70 with the compound; and (b) determiningwhether the compound restores, in whole or in part, a normal Ku70phenotype.

BRIEF DESCRIPTION OF THE FIGURES

[0024]FIG. 1

[0025] Inactivation of Ku70 by homologous recombination. (A)Diagrammatic representation of the Ku70 locus (top), the targetingconstruct (middle), and the targeted allele and hybridization probe(bottom). EcoRI restriction sites used to detect the targeted gene areindicated (21). (B) Southern blot of EcoRI-digested tail DNA fromcontrol wild type (WT), heterozygous (^(+/−)) and homozygous (^(−/−))Ku70-targeted mice. The wild-type and mutant fragments are 13 and 5.7 kbrespectively. (C) Western blot analysis showing that Ku70 protein is notexpressed in Ku70^(−/−) cells. Whole-cell lysates prepared from mouseear fibroblasts (50 μg) and mouse embryo fibroblasts (100 μg) wereseparated by 10% SDS-PAGE, transferred to a nitrocellulose membrane, andprobed with polyclonal antibodies against full-length rodent Ku80 (top)and Ku70 (bottom), respectively. (D) Gel mobility shift assay (22)showing the lack of DNA-end binding activity in Ku70^(−/−) cells. Ku-DNAbinding complex is indicated by arrow on the right.

[0026]FIG. 2

[0027] Development of B lymphocyte, but not T lymphocyte, is blocked atan early stage in Ku70^(−/−) mice. (A) Histology of thymus (Thy), lymphnodes (LN) and spleens (Spl) from wild type control mice, Ku70^(−/−)mice, and Ku80^(−/−) mice (23). Cortex (C) and medulla (M) areindicated. W, white pulp; R, red pulp; GC, germinal center. Panels a toi, tissue sections were stained with haematoxylin and eosin (HE); panelsj to l, tissue sections were stained with anti-CD3 (CD3); and panels mto o, tissues were stained with anti-CD19 (CD19). Anti-CD3 and anti-CD19antibodies were tested in both frozen and paraffin sections of wild-typelymphoid organs and showed the expected specific patterns of staining.(B) Flow cytometric analysis of thymocytes (Thy) bone marrow (BM) andspleen (Spl) cells from Ku70^(−/−) mice, Ku70^(+/+) littermates, andKu80^(−/−) mice. CD4, anti-CD4 monoclonal antibody; CD8, anti-CD8monoclonal antibody; B220, anti-B220 monoclonal antibody; CD43,anti-CD43 monoclonal antibody; IgM, anti-lgμ-heavy-chain monoclonalantibody. The data were gated for live lymphoid cells based on forwardand side scatter properties; 10,000-20,000 cells were analyzed persample. (C) Analysis of TCRβ chain expression in Ku70^(−/−) mice.Thymocytes and spleen cells were obtained from Ku70^(−/−), Ku80^(−/−),and wild type littermates and analyzed for expression of CD4, CD8 andTCRβ by 3-color flow cytometry. The TCRβ expression of both CD4+ andCD8+ single-positive T cells were shown.

[0028]FIG. 3

[0029] T-cell antigen receptor and immunoglobulin gene rearrangement inKu70^(−/−) mice. (A) Recombination of V558L, V7183 to DJ_(H), and D_(H)to J_(H) gene segments (26). 100 ng DNA was used for Ku70^(−/−) (lanes 7and 8), Ku80^(−/−) (lanes 1, 2, and 3), and SCID mice (lanes 4, 5, and6), and 1, 10 and 100 ng for WT mice (lanes 9-11). For IVS controls, DNAwas diluted 100-fold before PCR. (B) PCR analysis of TCR generearrangements. Thymus DNA was assayed for recombination of Vβ8-Jβ2 andDδ2 to Jδ1 rearrangements (20, 27, 28). 100 ng DNA was used forKu70^(−/−) (lanes 2 and 7), Ku80^(−/−) (lane 1), and Ku70^(−/−) mice(lane 7) and 1, 10 and 100 ng for WT mice (lanes 4-6). Controls includea 1-kb germline interval amplified in the Dδ2 to Jδ1 intervening region(germline) , and a non-recombining segment of the Ig locus between J_(H)and C_(H)1. The same thymus DNA samples were examined for Vβ8-Jβ2 andDδ2 to Jδ1 recombination. Abbreviations: DJ_(H), _(H)D to _(H)Jrearrangements; V7183J_(H) and V558LJ_(H), V7183 and V558L to DJ_(H)rearrangements (26); Vβ8Jβ2.1 to Vβ8-Jβ2.6, Vβ8 to DJβ2 rearrangements(28); germline, unrecombined DNA from the Dδ2 to Jδ1 interval; Dδ2Jδ1,Dδ2 to Jδ1 rearrangements (20, 27); IVS, non-recombining segment of theIg locus between J_(H) and C_(H)1 (26). Multiple lanes underneath eachgenotype label (Ku70^(−/−), Ku80^(−/−), and SCID) represent differentindividual animals.

[0030]FIG. 4

[0031] Disruption of Ku70 confers radiation hypersensitivity and adeficiency in DNA DSB repair. (A) Radiation survival curves for thegranulocyte/macrophage colony-forming units (CFU-GM) in the bone marrowof wild type (WT) , Ku70^(−/−), and Ku80^(−/−) mice (30, 32). (B)Deficiency in the repair of radiation-induced DSB in Ku70^(−/−) andKu80^(−/−) cells (31). Upper panel shows rejoining of DNA DSB producedby 40 Gy X-ray; (C) Induction of DNA DSB as a function of the radiationdose in WT, Ku70^(−/−) and Ku80^(−/−) cells. Symbols are • for WT, ▴ forKu70^(−/−), and ▪ for Ku80^(−/−) cells, respectively.

[0032]FIG. 5

[0033] Disruption of the Ku70 locus in mouse ES cells and generation ofKu70^(−/−) mice. (A) Diagrammatic representation of the Ku70 locus(top), the targeting construct (middle), the targeted allele (bottom)and the PCR primers. EcoRI (E) restriction sites used to detect thetargeted genes are indicated. (B) PCR analysis of tail DNA fromKu70^(+/+), Ku70^(+/−), and Ku70^(−/−) mice. The wild type sequencewhich was amplified using HO-4/HO-3 primers was not present inKu70^(−/−) mouse tail while the disrupted sequence primed by HO-4/HO-2was not expressed in Ku70^(+/+) mouse. (C) Postnatal growth ofKu70^(+/+) and Ku70^(−/−) littermates. Average weights of seven animalsfrom each genotype are plotted against time. There was no significantdifference in the body weight between Ku70^(+/+) and Ku70^(+/−) mice.(D) Photograph of 5-week-old Ku70^(+/+) and Ku70^(−/−) littermates.

[0034]FIG. 6

[0035] Survival curves of Ku70^(+/+), Ku70^(+/−), and Ku70^(−/−) mice.Sample sizes used for the statistical analysis (Kaplan and Meier, 1958)are: n (+/+)=102, n (+/−)=326, and n (−/−)=185.

[0036]FIG. 7

[0037] Histological analysis of the spontaneous tumors that developed inKu70^(−/−) mice. (A & D) Photomicrographs of sections of a thymiclymphoma processed as follows: (A), hematoxylin and eosin staining; (D),positive immunohistochemical surface staining against T-cell surfacemarker CD3. (B, C, E and F) Photomicrographs of sections of lung tissuesshowing tumor involvement. (B) and (C), hematoxylin and eosin; (E) and(F), positive immunohistochemical surface staining against T-cellsurface marker CD3. B, bronchial lumen; V, blood vessel. (G) Flowcytometric analysis of tumor cells. Cells were labeled withPE-conjugated anti-CD4 and FITC-conjugated anti-CD8 antibodies. Originalmagnifications: A, C, D and F, 400×; B and E, 100×.

[0038]FIG. 8

[0039] Neoplastic transformation of Ku70^(−/−) early-passage mouse earfibroblasts (MEFs). (A) Focus-formation assay. (B) Morphology oftransformed foci (type III). (C) Colony-formation assay in soft agar.Left, wild type (Ku70^(+/+)) MEFs untransformed; middle left, Ku70^(−/−)MEFs untransformed; middle right (focus T1), cells from a focus producedby spontaneous transformation of Ku70^(−/−) MEFs (passage 7); and right(focus C2), cells from a focus produced by transformation of E6/E7co-transfected Ku70^(−/−) MEFs. Cells from other randomly chosen fociwere also able to produce colonies in soft agar.

[0040]FIG. 9

[0041] Radiation sensitivity of Ku70^(−/−) fibroblasts and Ku70^(−/−)mice. (A) Ku70^(−/−) and wild-type Ku70^(+/+) primary ear fibroblasts(passage 7) were exposed to graded doses of Y-irradiation.Ku70-deficient cells show significantly decreased ability to formcolonies after ionizing radiation as compared with the wild-type cells.(B) Survival of Ku70^(−/−) and wild-type mice irradiated with 400 cGy.Five adult mice (4 months old) from each genotype were irradiatedsimultaneously and monitored for 2 weeks. Whereas all of the wild-typemice survived, 100% of the Ku70^(−/−) mice died within this period.

[0042]FIG. 10

[0043] Histological appearance of segmental gastrointestinalabnormalities of Ku70^(−/−) mice. Gastrointestinal tissues from athree-month-old Ku70^(−/−) mouse were stained with hematoxylin and eosinand photographed. (A) Normal appearance of the intestine showing thepresence of ganglions (400×). (B) Section of intestine from the sameanimal showing absence of ganglion neurons (400×). (C) At a lowermagnification (100×) segmental aganglionosis that developed in aKu70^(−/−) mouse is demonstrated. The left portion of the specimen showscomplete absence of ganglion neurons. This phenotype is associated withthe effacement of the typical morphology of the intestinal villi,dilation of intestinal lumen, and denudation of the mucosa, as well assegmental distention of the intestine. In contrast, the right portion ofthe specimen shows a normal appearance as observed in the wild-typelittermates.

[0044]FIG. 11

[0045] Ku70 alteration in human tumors. Immunohistochemical analysis ofKu70 expression in human T-cell lymphomas. (A-C), B-cell lymphomas (D-F)and in human normal spleen (G). The photomicrograph of the spleen(paraffin) illustrates the nuclear staining against Ku70 (G). (A)Photomicrograph illustrating a T-cell lymphoma (sample #T2—paraffin)with positive nuclear staining against Ku70, (B and C) Photomicrographsof T-cell lymphomas (samples #T13 and T9—paraffin and frozen,respectively) showing negative immunohistochemical staining againstKu70. In panel (C), the arrows point to endothelial cells with positivenuclear staining for Ku70, which served as internal positive controls.(D) Photomicrograph illustrating a B-cell lymphoma (sample #B4—paraffin)with positive nuclear staining against Ku70. (E) Photomicrograph of aB-cell lymphoma (sample #B8—paraffin) showing negativeimmunohistochemical staining against Ku70. (F) Photomicrograph of aB-cell lymphoma (sample #B9—frozen) showing cytoplasmic staining ofKu70. Original magnification: A to G, 400×. (H) Representative PCR-SSCPanalysis. Lane 3 illustrates the Ku70 band shift identified by PCR-SSCPcorresponding to sample #T3. Lane 1, internal control (normal); lane 2,tumor corresponding to sample #T8, showing no band shift. Directsequencing results of the PCR product obtained from tumor sample #T3 areshown below. The single base pair substitution (ACA→ATA) was found to betumor-specific (absent in normal tissue) affecting codon 292, changing athreonine to isoleucine. (I) Representative RT-PCR direct sequencingfrom a T-cell lymphoma (sample #T3) and its corresponding normal tissue.Single base substitutions are indicated at codons 452 (ATC-GTC) and 453(ATG-ACG), changing isoleucine to valine and methionine to threonine,respectively. These alterations were found to be tumor-specific and wereabsent in normal tissue. (J) Representative RT-PCR direct sequencingfrom a neuroblastoma (sample #N10) and its corresponding normal tissue.Single base substitutions are indicated at codon 530 (TAC→CAC) and codon529 (GTT→GTC), changing tyrosine to histidine at codon 530, andproducing a silent mutation at codon 529 (valine to valine),respectively. These mutations were also found to be tumor-specific andwere absent in corresponding normal tissue.

[0046]FIG. 12

[0047] Effect of (A) radiation, (B) bleomycin, (C) Adriamycin, and (D)Etoposide on Ku70 and Ku80 deficient mouse cells.

[0048]FIG. 13

[0049] Effect of (A) radiation, and (B) adriamycin on different celltypes. o=HeLa controls cells; ▪=HeLa cells expressing antisense Ku70;▴=HeLa cell expressing antisense Ku80.

[0050]FIG. 14

[0051] Nucleotide sequences of Vβ8Dβ2.1Jβ2.6 junctions from the thymusof a 4 week old Ku70^(−/−) mouse. Products corresponding to Vβ8.1, Vβ8.2or Vβ8.3 rearrangement with Jβ2.6 were cloned and sequenced. TCR Vβ8-Jβ2joints were amplified by PCR (20, 27, 28) as described (see FIG. 3B).PCR cycling conditions were 94° C. for 45″, 68° C. for 30″, and 72° C.for 30″ (30 cycles). The band corresponding to Vβ8-Jβ2.6 was purified,reamplified for 20 cycles and then subcloned into the PCRII vector(Invitrogen). DNA was extracted from individual colonies and sequencedusing the universal T7 and M13 reverse primers. Germline sequences arewritten in bold case, ‘N’ and ‘P’ denote nucleotides not present in thegermline sequences.

DETAILED DESCRIPTION OF THE INVENTION

[0052] This invention provides a method of diagnosing a predispositionto cancer in a subject comprising: (a) obtaining a nucleic acid samplefrom the subject; and; (b) determining whether one or more of thesubject's Ku70 alleles or regulatory regions to those alleles aredeleted or different from the wild type so as to reduce or eliminate thesubject's expression of polypeptide having tumor suppressor activity.

[0053] Nucleic acid samples can be obtained from numerous sources whichinclude, but are not limited to, blood, saliva, tissue, and hairfollicles.

[0054] Ku70 allele and regulatory element differences from the wild typeinclude, but are not limited to, deletions, additions, substitutions,and chemical modification of Ku70. Chemical modifications of Ku70 and/orits regulatory elements include, but are not limited to, nucleotidephosphorylation, methylation, and/or hydroxylation. Sites of nucleotidedifferences include, but are not limited to, introns, exons, enhancers,promoters, splice junctions, splice site consensus sequences,RNA-cleavage/polyadenylation sites, cap sites, and other protein bindingsites. In an embodiment, protein binding sites are located by“footprinting”, wherein a protein shields the nucleic acid from cleavageby restriction enzymes.

[0055] Ku70 allele or regulatory differences also include, but are notlimited to, sequence changes that alter the relative distance of theKu70 allele from its regulatory elements. In an embodiment, an insertionincreases the distance of Ku70 from an enhancer. In another embodiment,an deletion, increase the proximity of Ku70 to its promoter.

[0056] Ku70 regulatory differences further include hypermethylation ofregulatory regions. In an embodiment, the promoter to Ku70 ishypermethylated. In another embodiment, the enhancer to Ku70 ishypermethylated.

[0057] In an embodiment, hypermethylation is associated with regions ofDNA that have reduced activity or no activity. In an embodimentmethylation of Ku70 promoter sequences is likely to reduce transciptionof Ku70. In an embodiment, methylation of other regions of Ku70, besidesthe promoter, including the coding region would also be indicative ofreduced Ku70 activity. In an embodiment methylation of Ku70 or itsregulatory regions occurs at CG pairs. In another embodiment,methylation of Ku70 or its regulatory regions occurs at sites containinga high a frequency of CG pairs.

[0058] In an embodiment, methylation is assessed by using restrictionendonucleases whose recognition sequences contain CG. For example, HpaIIand MspI both recognize CCGG. However, HspII cleaves only unmethylatedDNA while MspI cleaves both methylated and unmethylated DNA. Using thesetwo restriction enzymes, one skilled in the art could infer from thesize of the restriction fragments the presence of methylation in Ku70 orits regulatory elements.

[0059] This invention also provides the above-described method, whereinthe cancer is T-cell lymphoma.

[0060] This invention further provides the above-described methods,wherein the cancer is B-cell lymphoma.

[0061] This invention also provides the above-described methods, whereinthe cancer is neuroblastoma.

[0062] In addition, this invention provides the above-described methods,wherein the regulatory region is a promoter.

[0063] This invention further provides the above-described methods,wherein the determining of step b comprises generating a polypeptideencoded by one or more of the subject's Ku70 alleles and comparing theresulting polypeptide to a wild type Ku70 polypeptide.

[0064] In an embodiment, comparing a polypeptide to Ku70 can be done bycomparing the polypeptide with wild type Ku70 for Ku70-type activity.Significant differences may result in Ku70 defect symptoms, whichinclude, but are not limited to, impaired DNA double-strand breakrepair, impaired V(D)J recombination, proportional dwarfism, increasedsister chromatid exchange, and hypersensitivity to ionizing radiation.Given Ku70's role DNA double strand break repair other functional assayswould be readily apparant to one skilled in the art.

[0065] In another embodiment, comparing a polypeptide to Ku70 can bedone by comparing the polypeptide sequences. One skilled in the artwould be able recognize sequence differences and chemical modificationsthat would influence the polypeptides activity. For example,substitutions that change the charge of amino acids in importantfunctional domains are likely to influence Ku70 activity.

[0066] In a different embodiment, comparing a polypeptide to Ku70 can bedone by testing the polypeptides for immunoreactivity. The ability togenerate and react with antibodies is indicative of the structuralproperties of the polypeptide. For example, a mutation thatsignificantly changes the functional properties of the polypeptide mayalso alter the polypeptides structural shape (i.e. conformation) in amanner that may mask or unmask various epitopes. Such structural changesmay be detected by antibodies that recognize epitopes in regionsinfluence by the structural changes.

[0067] This invention also provides the above-described methods, whereinthe nucleic acid sample is obtained from the subject's blood.

[0068] This invention further provides a method of diagnosing apredisposition to cancer in a subject comprising: determining the levelof Ku70 expression in the subject.

[0069] In addition, this invention provides the above-described methods,wherein the cancer is T-cell lymphoma.

[0070] This invention also provides the above-described methods, whereinthe cancer is B-cell lymphoma.

[0071] In addition, this invention provides the above-described methods,wherein the cancer is neuroblastoma.

[0072] This invention provides the above-described methods, wherein thelevel of Ku70 expression is determined based upon the level of Ku70 mRNAin the subject.

[0073] This invention also provides the above-described methods, whereinthe level of Ku70 expression is determined based upon the level of Ku70polypeptide in the subject.

[0074] This invention further provides the above-described methods,wherein zero or reduced Ku70 expression indicates a predisposition tocancer.

[0075] In addition, this invention provides a method of diagnosing apredisposition to cancer in a subject comprising: determining thesubcellular localization of Ku70 in the subject, wherein an abnormalsubcellular localization of Ku70 indicates a predisposition to cancer.

[0076] This invention also provides the above-described methods, whereinthe abnormal subcellular localization of Ku70 comprises increasedcytosolic localization of Ku70.

[0077] This invention further provides the above-described methods,wherein the abnormal subcellular localization of Ku70 comprisesdecreased nuclear localization of Ku70.

[0078] This invention also provides a method of assessing the severityof cancer in a subject comprising: (a) obtaining a nucleic acid samplefrom the subject; and (b) determining whether one or more of thesubject's Ku70 alleles or regulatory regions to those alleles aredeleted or different from the wild type so as to reduce or eliminate thesubject's expression of polypeptide having tumor suppressor activity.

[0079] In addition, this invention provides the above-described methods,wherein the cancer is T-cell lymphoma.

[0080] This invention further provides the above-described methods,wherein the cancer is B-cell lymphoma.

[0081] This invention further provides the above-described methods,wherein the cancer is neuroblastoma.

[0082] This invention also provides a method of assessing the severityof cancer in a subject comprising: determining the subcellularlocalization of Ku70 in the subject, wherein an abnormal subcellularlocalization of Ku70 indicates a predisposition to cancer.

[0083] In addition, this invention provides the above-described methods,wherein the abnormal subcellular localization of Ku70 comprisesincreased cytosolic localization of Ku70.

[0084] This invention also provides the above-described methods, whereinthe abnormal subcellular localization of Ku70 comprises decreasednuclear localization of Ku70.

[0085] Further, this invention provides a method of inhibiting thegrowth of cancer cells, comprising introducing into a cell a Ku70 geneunder conditions permitting expression of the gene.

[0086] In addition, this invention provides a method of inhibitingcancer, comprising introducing into a cell a Ku70 gene under conditionspermitting expression of the gene.

[0087] This invention also provides the above-described methods, whereinthe cancer is T-cell lymphoma.

[0088] As used herein, inhibiting T-cell lymphoma includes, reducing thelikelihood of or preventing the occurrence of T-cell lymphoma in asubject who does not have T-cell lymphoma. Inhibiting T-cell lymphomafurther includes, reducing or eliminating the occurrence of T-celllymphoma in a subject who does have T-cell lymphoma.

[0089] This invention further provides a method of inhibiting the growthof cancer cells, comprising introducing into a cell a Ku70 gene underconditions permitting expression of the gene.

[0090] This invention provides the above-described methods, wherein thecancer is B-cell lymphoma.

[0091] This invention further provides the above-described methods,wherein the cancer is neuroblastoma.

[0092] In addition, this invention provides the above-described methods,wherein the cell prior to the introduction of the Ku70 gene wascharacterized as having a mutation at one or more Ku70 alleles orregulatory regions thereto.

[0093] This invention also provides the above-described methods, whereinthe mutation is a frameshift mutation.

[0094] This invention provides the above-described methods, wherein themutation is a point mutation.

[0095] In addition, this invention provides the above-described methods,wherein the cell prior to the introduction of the Ku70 gene wascharacterized as having reduced expression of Ku70.

[0096] This invention further provides the above-described methods,wherein the Ku70 gene is incorporated into an expression vector prior tointroduction into the cell.

[0097] Numerous vectors for expressing the inventive proteins may beemployed. Such vectors, including plasmid vectors, cosmid vectors,bacteriophage vectors and other viruses, are well known in the art. Forexample, one class of vectors utilizes DNA elements which are derivedfrom animal viruses such as bovine papilloma virus, polyoma virus,adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV orMoMLV), Semliki Forest virus or SV40 virus. Additionally, cells whichhave stably integrated the DNA into their chromosomes may be selected byintroducing one or more markers which allow for the selection oftransfected host cells. The markers may provide, for example,prototrophy to an auxotrophic host, biocide resistance or resistance toheavy metals such as copper. The selectable marker gene can be eitherdirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation.

[0098] Regulatory elements required for expression include promotersequences to bind RNA polymerase and transcription initiation sequencesfor ribosome binding. Additional elements may also be needed for optimalsynthesis of mRNA. These additional elements may include splice signals,as well as enhancers and termination signals. For example, a bacterialexpression vector includes a promoter such as the lac promoter and fortranscription initiation the Shine-Dalgarno sequence and the start codonAUG. Similarly, a eukaryotic expression vector includes a heterologousor homologous promoter for RNA polymerase II, a downstreampolyadenylation signal, the start codon AUG, and a termination codon fordetachment of the ribosome. Such vectors may be obtained commercially orassembled from the sequences described by methods well known in the art,for example the methods described above for constructing vectors ingeneral.

[0099] These vectors may be introduced into a suitable host cell to forma host vector system for producing the inventive proteins. Methods ofmaking host vector systems are well known to those skilled in the art.

[0100] Suitable host cells include, but are not limited to, bacterialcells (including gram positive cells), yeast cells, fungal cells, insectcells and animal cells. Suitable animal cells include, but are notlimited to HeLa cells, Cos cells, CV1 cells and various primarymammalian cells. Numerous mammalian cells may be used as hosts,including, but not limited to, the mouse fibroblast cell NIH-3T3 cells,CHO cells, HeLa cells, Ltk⁻ cells and COS cells. Mammalian cells may betransfected by methods well known in the art such as calcium phosphateprecipitation, electroporation and microinjection.

[0101] In an embodiment, inducible promoters may be fused with thecoding region of the DNA to provide an experimental means to regulateexpression. Alternatively or in addition, tissue specific regulatoryelements may be fused with the coding region to permit tissue-specificexpression.

[0102] This invention also provides a method of inhibiting the growth ofcancer cells, comprising introducing Ku70 into a cell.

[0103] This invention also provides a method of inhibiting cancer,comprising introducing Ku70 into a cell.

[0104] In addition, this invention provides the above-described methods,wherein the cancer is T-cell lymphoma.

[0105] This invention also provides the above-described methods, whereinthe cancer is B-cell lymphoma.

[0106] This invention further provides the above-described methods,wherein the cancer is neuroblastoma.

[0107] In addition, this invention provides a transgenic cell, whereinthe expression of the Ku70 allele has been altered to increase thesusceptibility of the cell to DNA damage.

[0108] This invention also provides a transgenic cell, wherein theexpression of the Ku70 allele has been altered to increase thesusceptibility of the cell to cancerous growth.

[0109] This invention also provides a transgenic organism, comprising anorganism whose germ line cells has been altered at the Ku70 allele toproduce an organism whose offspring have an increased likelihood ofdeveloping tumors.

[0110] Germ line cells include, but are not limited to, spermatogonium,primary spermatocytes, secondary spermatocytes, spermatids, sperm cells,oogonium, primary oocytes, second oocytes, and egg cells.

[0111] Methods to introduce a nucleic acid into cells have been wellknown in the art. Naked nucleic acid may be introduced into the cell bydirect transformation. Alternatively, the nucleic acid molecule may beembedded in liposomes. Accordingly, this invention provides the abovemethods wherein the nucleic acid is introduced into the cells by nakedDNA technology, adenovirus vector, adeno-associated virus vector,Epstein-Barr virus vector, Herpes virus vector, attenuated HIV vector,retroviral vectors, vaccinia virus vector, liposomes, antibody-coatedliposomes, calcium phosphate coprecipitation, mechanical or electricalmeans (i.e. electroporation).

[0112] Transgenic organisms may be created by a variety of techniques.One such technique is introducing normal or mutant genes into fertilizedembryos. In an embodiment, the gene is cloned and then microinjectedinto the pronuclei of a fertilized egg.

[0113] One example of producing a transgenic animal is as follows:female mice are mated, and the resulting fertilized eggs are dissectedout of their oviducts. The eggs are stored in an appropriate medium suchas M2 medium. DNA or cDNA encoding a Ku70 is purified from a vector bymethods well known in the art. Inducible promoters may be fused with thecoding region of the DNA to provide an experimental means to regulateexpression of the transgene. Alternatively or in addition, tissuespecific regulatory elements may be fused with the coding region topermit tissue-specific expression of the transgene. The DNA, in anappropriately buffered solution, is put into a microinjection needle(which may be made from capillary tubing using a pipet puller) and theegg to be injected is put in a depression slide. The needle is insertedinto the pronucleus of the egg, and the DNA solution is injected. Theinjected egg is then transferred into the oviduct of a pseudopregnantmouse (a mouse stimulated by the appropriate hormones to maintainpregnancy but which is not actually pregnant), where it proceeds to theuterus, implants, and develops to term. Microinjection is not the onlymethod for inserting DNA into the egg cell, and is used here only forexemplary purposes.

[0114] Another technique for generating a transgenic organism isintroducing the DNA into embryonic stem cells and then injecting thetransfected stem cells into embryos where they may become incorporatedinto the developing embryo. This technique has the advantage of creatingthe opportunity for screening and selecting particular transfected stemcells prior to their incorporation into the embryo. In an embodiment,antibiotic resistance may be used to select for stem cells that havetaken up the DNA of interest. In another embodiment, PCR may be used toscreen for stems cell that have incorporated only one copy of thetransgene. In another embodiment, PCR and/or sequencing can be used toscreen for stem cells that have incorporated the transgene throughhomologous recombination.

[0115] When DNA is introduced into a cell, the incorporation of the DNAinto the cell's genome is dependent on recombination events.Recombination is of two types: heterologous recombination, wherein theDNA recombines into an unrelated sequence; and homologous recombination,wherein the DNA recombines into an identical or allelic variant of thesequence in the genome. Homologous recombination is common in bacteria,yeast, and certain viruses, but is rare in mammalian cells. Usinghomologous recombination, it is possible to create organisms in whichthe function of the native gene has been “knocked out.” By introducingthe DNA into embryonic stem cells, it is possible to select or screenfor specific cell types for introduction into embryos. For example, PCRmay be used to screen for homologous recombination events in embryonicstem cells. By selecting for such events and introducing the stem cellsinto mouse embyros, “knockout mice”, may be created in which specificgenes have been replaced by different nucleic acid sequences that do notencode the native protein.

[0116] Prior to its incorporation into the cellular DNA, the DNAfragments to be introduced often become ligated end-to-end by enzymes inthe cell, thus forming long tandem arrays. Because of ligation,transgenic organisms may contain several copies of the transgene.

[0117] Chimeric transgenic organisms may result from the above-describedtechniques. For example, the embryonic stems cells introduced into ablastocyst may codevelop with the other embryonic cells native to theblastocyst. The resulting organism may have somatic cells that differ intheir genetic content. In an example, a transgenic rabbit may havedifferent colored patches of fur resulting from a coat color transgene.If the germ line cells of a transgenic organism have incorporated thetransgene, then the offspring of the transgenic organism may alsoincorporate the transgene. In an embodiment, chimeric transgenicorganisms whose germ line cells have incorporated the transgene may bebred together to produce transgenic organisms that have the transgenethroughout their cells.

[0118] In addition, this invention provides a transgenic organism,comprising an organism whose germ line cells has been altered at theKu70 allele to produce an organism whose offspring have an increasedlikelihood of having increased susceptibility to DNA damage.

[0119] This invention provides the above-described transgenic organisms,wherein the somatic cells have been altered to reduce or eliminateexpression of Ku70.

[0120] This invention also provides the above-described transgenicorganisms, wherein the somatic cells have been alterred to reduce oreliminate expression of Ku70.

[0121] This invention also provides the above-described transgenicorganisms, wherein the organism is a mouse.

[0122] This invention provides the above-described transgenic organisms,wherein the organism is a mouse.

[0123] This invention further provides a method of screening a compoundfor carcinogenic activity, comprising: (a) contacting cells havingreduced expression of Ku70 with the compound; and (b) determiningwhether the compound results in a malignant transformation phenotype.

[0124] A malignant transformation phenotype includes but is not limitedto metastasis and/or anchorage independent growth.

[0125] This invention also provides the above-described methods, whereinthe cell is a fibroblast.

[0126] In addition, this invention provides the above-described methods,wherein the malignant transformation phenotype comprises anchorageindependent growth.

[0127] This invention provides a method of screening a compound for DNAdamaging activity, comprising: (a) contacting cells having reducedexpression of Ku70 with the compound; and (b) determining whether thecompound results in DNA damage.

[0128] This invention also provides the above-described methods, whereinthe DNA damage is determined by measuring a reduction in cell survival.

[0129] In addition, this invention provides the above-described methods,wherein the DNA damage comprises one or more double strand breaks.

[0130] This invention also provides a method of screening a compound forability to restore Ku70 activity to cells having Ku70 defect symptomsresulting from reduced Ku70 activity, comprising: (a) contacting cellshaving reduced expression of Ku70 with the compound; and (b) determiningwhether the compound restores, in whole or in part, a normal Ku70phenotype.

[0131] This invention will be better understood from the examples whichfollow. However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention as described more fully in the claims which follow thereafter.

[0132] Experimental Details

[0133] First Series of Experiments

[0134] Material and Methods

[0135] Target Disruption of Ku70 and Generation of Ku70^(−/−) Mice

[0136] Mouse genomic Ku7o gene was isolated from a sCos-I cosmid libraryconstructed from a mouse strain 129 embryonic stem cell lines (21). Thereplacement vector was constructed using a 1.5 kb 5′-fragment whichcontains the promoter locus with four GC-box and exon 1, and a 8 kbEcoRV-EcoRI fragment extending from intron 2 to intron 5 as indicated inFIG. 1a. Homologous replacement results in a deletion of 336-bp exon 2including the translational initiation codon.

[0137] The targeting vector was linearized with NotI and transfectedinto CJ7 embryonic stem (ES) cells by electroporation using a Bio-RadGene Pulser. Three hundred ES cell clones were screened, and 5 clonescarrying the mutation in Ku70 were identified by Southern blotting.Positive ES clones were injected separately into C57BL/6 blastocysts togenerate chimeric mice. One clone was successfully transmitted throughthe germline after chimeras were crossed with C57 BL/6 females.Homozygous Ku70^(−/−) mice were generated by crossing Ku70^(+/−)heterozygotes.

[0138] The genotype of the mice was first determined by tail PCRanalysis which distinguishes endogenous from the targeted Ku70 allele,and subsequently confirmed by Southern blot analysis. The PCR reactioncontained 1 μg genomic DNA; 0.6 μM (each) of primers HO-2:GGGCCAGCTCATTCCTCCACTCATG, HO-3: CCTACAGTGTACCCGGACCTATGCC and HO-4:CGGAACAGGACTG-GTGGTTGAGCC; 0.2 mM (each) DNTP; 1.5 mM MgCl₂ and 2.5 U ofTaq polymerase. Cycling conditions were 94° C. for 1 min, 64° C. for 1min, 72° C. for 1 min (30 cycles), followed by an extension at 72° C.for 10 min. Primers HO-2 and HO-4 give a product of the targeted allelethat is −380 bp; primers HO-3 and HO-4 yield a wild type product of 407bp.

[0139] Western Blot Analysis and Gel Mobility Shift Assay

[0140] To confirm that the disruption of Ku70 produces a null mutation,Ku70 protein expression was measured by Western blotting usingpolyclonal antisera against intact mouse Ku70. The lack of Ku70 was alsoverified by a Ku-DNA-end binding assay (gel mobility shift analysis).Cell extracts were prepared and gel mobility shift assays were performedas described (22). Equal amounts of cellular protein (50 μg) fromKu70^(+/+) (WT), Ku70^(+/−), and Ku70^(−/−) mouse embryo fibroblastswere incubated with a ³²P-labeled double-stranded oligonucleotide,5′-GGGCCAAGAATCTTCCAGCAGTTTCGGG-3′. The protein-bound and freeoligonucleotides were electrophoretically separated on a 4.5% nativepolyacrylamide gel. Gel slabs are dried and autoradiographed with KodakX-Omat film.

[0141] Immunohistochemistry

[0142] To determine the pathological changes, histological sections ofvarious organs of Ku70^(−/−), Ku80^(−/−) and wild type littermate micewere prepared and examined as previously described (23). Lymph nodes,spleens and thymuses from 4- to 5-week-old mice were fixed in 10%buffered formalin and embedded in paraffin, or embedded in OCT compound(Miles Laboratories) and frozen in liquid nitrogen at −70° C. Sections(5 μm) were stained with hematoxylin and eosin, and representativesamples were selected for immunohistochemical analysis.Immunophenotyping was performed using an avidin-biotin immunoperoxidasetechnique (24). Primary antibodies included anti-CD3 (purified rabbitserum, 1:1000, Dako), anti-B220 (rat monoclonal, 1:1000, Pharmingen)anti-CD19 (rat monoclonal, 1:1000, Pharmingen), and were incubatedovernight at 4° C. Samples were subsequently incubated with biotinylatedsecondary antibodies (Vector Laboratories) for 30 min (goat anti-rabbit,1:100; rabbit anti-rat, 1:100), and then with avidin-biotin peroxidase(1:25 dilution, Vector Laboratories) for 30 min. Diaminobenzadine wasused as the chromogen and hematoxylin as the counter stain. Wild typelymphoid organs including thymus, spleen and lymph nodes from differentmice were used for titration of the antibodies and positive controls.Anti-CD3 and anti-CD19 antibodies were tested in both frozen andparaffin sections of wild-type lymphoid organs and showed the expectedspecific patterns of staining. For negative controls, primary antibodieswere substituted with class-matched but unrelated antibodies at the samefinal working dilutions.

[0143] Cell Preparation and Flow Cytometric Analysis

[0144] For flow cytometry, single cell suspensions from lymphoid organsof 4- to 6-week-old mutant and littermate control mice were prepared forstaining as described previously (19) and analyzed on a Becton DickinsonFACs Scan with Cell Quest software. Cells were stained with combinationsof phycoerythrin-(PE) labeled anti-CD4, and fluorescein (FITC)-labeledanti-CD8, or PE labeled anti-B220, and FITC-labeled anti-CD43, or FITCanti-μ and PE anti-B220 (Pharmingen), as needed. Bone marrow cells wereharvested from femurs by syringe lavage, and cells from thymus andspleen were prepared by homogenization. Cells were collected and washedin PBS plus 5% FCS and counted using a hemacytometer. Samples fromindividual mice were analyzed separately. Dead cells were gated out byforward and side scatter properties. Experiments were performed at leastthree times and yielded consistent results.

[0145] DNA Preparation and Analysis of V(D)J Recombination Products

[0146] To determine whether a null mutation in Ku70 affects therecombination of antigen-receptor genes in T and B lymphocytes in vivo,we measured the immunoglobulin and T-cell antigen receptor (TCR)rearrangements by PCR. DNA from bone marrow was amplified with primersspecific to immunoglobulin D-J_(H) and V-DJ_(H) rearrangements, and DNAfrom thymus was amplified with primers that detect V-DJ_(β) andD_(δ)-J_(δ)-rearrangement (20, 25-28). Oligonucleotides for probes andPCR primers specific to TCR Vβ-Jβ rearrangements and immunoglobulinD-J_(H) and V_(H)-DJ rearrangements are as follows. For TCRβ8-Jβ2rearrangements (28): Vβ8.1: 5′-GAGGAAAGGT-GACATTGAGC-3′, Jβ2.6:5′-GCCTGGTGCCGGGACCGAAGTA-3′, Vβ8 probe: 5′-GGGCTG AGGCTG ATCCATTA-3′.For D_(δ2)-J_(δ1) rearrangement (20, 27): DR6:5′-TGGCTTGACATGCAGAAAACACCTG-3′, DR53: 5′-TGAATTCCACAG-TCACTTGGCTTC-3′,and DR2 probe: 5′-GACACGTGATACAAAGCCCAGGGAA-3′. For immunoglobulinD-J_(H) and V-DJ_(H) rearrangements (26): 5′D:5′-GTCAAGGGATCTACTACTGTG-3′, V7183:5′-GAGAGAATTCAGAGACAATC-CCAAGAACACCCTG-3′, VJ558L:5′-GAGAGAATTCTCCTCCAGCACAG-CCTACATG-3′, J2:5′-GAGAGAATTCGGCTCCCAATGACCCTTTCTG-3′, 5′IVS:5′-GTAAGAATGGCCTCTCCAGGT-3′, 3′-IVS: 5′-GACTCAATCACTAAGACA-GCT-3′, andprobe: a 6 kb EcoR I fragment covering the J region of mouse IgM.

[0147] Cell Survival Determination

[0148] 8- to 10-week-old Ku70^(−/−) and Ku80^(−/−) mice and wild typelittermates were used for our studies. Bone marrow cell suspensions wereprepared by flushing the femur with MEM supplemented with 15% fetal calfserum (FCS). The cell suspension was then counted using a hemacytometerand centrifuged at 1000 rpm for 12 min. The resulting pellet wasresuspended and diluted to approximately 1×10⁶ cells/ml in MEM plus 15%FCS for further experiments.

[0149] To measure the survival of granulocyte-macrophage progenitors,the method of Van Zant et al. (29) was used with minor modifications(30). Briefly, α-MEM contained 30% heat-inactivated FCS and 1% bovineserum albumin; in addition, 0.5 ng/ml GM-CSF (R & D Systems) was used asa source of colony-stimulating factor. One day before each experiment,2.0 ml of the above media containing 0.5% noble agar (DIFCOLaboratories) was added to individual 60-mm petri dishes. Immediatelyafter radiation exposure, cells were diluted in 2 ml of the above mediawith 0.3% noble agar and poured over the prepared dishes with 0.5% nobleagar underlayer. The cells were then incubated at 37° C. with 5% CO₂ and95 to 98% humidity. The colonies were counted on Day 8 with a dissectingmicroscope. Macrophage and granulocyte colonies were counted separatelyand then summed together for survival calculations ofgranulocyte-macrophage progenitors (CFU-GM). Only colonies containing 50or more cells were scored. The colony forming efficiency of CFU-GMs was60 to 100/10⁵ nucleated cells for untreated controls. Surviving fractionwas defined as the cloning efficiency of irradiated marrow cellsrelative to that of untreated controls. All experiments were performedat least twice and yielded consistent results.

[0150] Asymmetric Field Inversion Gel Electrophoresis

[0151] To determine the rate and extent of DNA DSB repair inKu-deficient cells after exposure to ionizing radiation, primary embryofibroblasts derived from Ku70^(−/−), Ku80^(−/−) and wild type littermatemice were used. Mouse embryo fibroblasts from 13.5-day embryos growingin replicate cultures for 3 days in the presence of 0.01 μCi/ml¹⁴C-thymidine (NEN) and 2.5 μM cold thymidine were exposed to 40 Gy ofX-rays and returned to 37° C. At various times thereafter, one dish wasremoved and trypsinized on ice; single cell suspensions were made andembedded in an agarose plug at a final concentration of 3×10⁶ cells/ml.AFIGE (Asymmetric Field Inversion Gel Electrophoresis) was carried outin 0.5% Seakem agarose (FMC, cast in the presence of 0.5 μg/ml ethidiumbromide) in 0.5× TBE (45 mM Tris, pH 8.2, 45 mM boric acid, 1 mM EDTA)at 10° C. for 40 h, by applying cycles of 1.25 V/cm for 900 sec in thedirection of DNA migration, and 5.0 V/cm for 75 sec in the reversedirection as described (31). Quantification and analysis for DNA DSBpresent were carried out in a PhosphorImager (Molecular Dynamics).Levels of DNA double-strand breaks (DSB) were quantified by calculatingthe FAR (fraction of activity released from the well into the lane) inirradiated and unirradiated samples, which equals the ratio of theradioactivity signal in the lane versus that of the entire sample (wellplus lane).

[0152] Experimental Results

[0153] Targeted Disruption of Ku70 Gene

[0154] To study the role of Ku70 in vivo, we generated mice containing agermline disruption of the Ku70 gene. Murine genomic Ku70 gene wasisolated and a targeting vector was constructed (FIG. 1a). Homologousreplacement results in a deletion of 336-bp exon 2 including thetranslational imitation codon. Two targeted ES clones carrying themutation in Ku70 were injected into C57BL/6 blastocysts to generatechimeric mice. One clone was successfully transmitted through thegermline after chimeras were crossed with C57BL/6 females. No obviousdefects were observed in Ku70^(+/−) heterozygotes, and these Ku70^(+/−)mice were subsequently used to generate Ku70^(−/−) mice (FIG. 1b). 25%of the offspring born from Ku70^(+/−)×Ku70^(+/−) crosses wereKu70^(−/−). Adult Ku70^(−/−) mice are fertile, but give reduced littersize (2 to 4 pups) as compared to the Ku70^(+/−) or Ku70^(+/+) mice(about 8 pups).

[0155] To confirm that the disruption produced a null mutation, Ku70protein expression was analyzed by both Western blotting (FIG. 1C) and aDNA end binding assay (FIG. 1D). Ku70 immunoreactivity was undetectable(FIG. 1C), and there was no Ku DNA-end binding activity in Ku70^(−/−)fibroblasts (FIG. 1D). The Ku80 subunit of the Ku heterodimer was found,but at much reduced levels (FIG. 1C), suggesting that the stability ofKu80 is compromised by the absence of Ku70. These observations areconsistent with the finding that the level of Ku70 was significantlyreduced in Ku80^(−/−) fibroblasts and Ku80^(−/−) ES cells (19). Takentogether, these data suggest that the stability of either component ofKu is compromised by the absence of the other.

[0156] Newborn Ku70^(−/−) mice were 40-60% smaller than their Ku70^(+/−)and Ku70^(+/+) littermates. During a 5-month observation period,Ku70^(−/−) mice grew and maintained body weight at 40-60% of controls.Thus Ku70^(−/−) mice, like Ku80^(−/−) mice are proportional dwarfs (19).

[0157] Development of B Lymphocyte, But Not T Lymphocyte, Is Blocked atEarly Stage in Ku70^(−/−) Mice

[0158] Examination of various organs from Ku70^(−/−) mice showedabnormalities only in the lymphoid system (FIG. 2A). Spleen and lymphnodes were disproportionately smaller by 5-10 fold relative to controls.In particular, splenic white pulp nodules were significantly reduced.Immunohistochemistry on deparaffinized tissue sections revealed that thesplenic white pulp contained cells that stained with anti-CD3 (i.e., CD3positive T cells), but there were no CD19 positive B cells (FIG. 2A,panels k and n). The Ku70^(−/−) thymus was also disproportionatelysmaller and contained 100-fold fewer lymphocytes than Ku70^(+/+)littermates (2×10⁶ in the former versus 2×10⁸ in the latter; measured in3 mice of each genotype). In contrast to the Ku80^(−/−) mice, theKu70^(−/−) thymus displayed normal appearing cortical-medullaryjunctions (FIG. 2A, panels g and j). Overall, the lymphoid tissues andorgans of Ku70^(−/−) mice are somewhat disorganized and much smallerthan Ku70^(+/+) mice (Table I); yet, they are relatively more developedand slightly larger than in Ku80^(−/−) mice. TABLE 1 LymphoidCellularity of Ku70−/− Mice Cell Cell Cell content content content (×1(×1 (×1 Tissue and million) million) million) geno type Total B220+ CD4+CD8+ Thymus wild type (n =4) 155 +/− 42  — 104 +/− 28  Ku70−/− (n = 3)2.98 +/− 0.91 — 0.6 +/− 0.2 Ku80−/− (n = 2) 1.0 +/− 0.5 — — Bone Marrowwild type (n = 4) 11.9 +/− 3.3  5.5 +/− 1.5 — Ku70−/− (n = 3) 7.2 +/−2.9 1.1 +/− 0.4 — Ku80−/− (n = 2) 9.0 +/− 3.0 — — Spleen wild type (n =4) 53 +/− 20 29 +/− 11 — Ku70−/− (n = 3) 6.5 +/− 1.3 0.4 +/− 0.2 —Ku80−/− (n = 2) 1.2 +/− 0.5 — —

[0159] To further examine the immunological defect in Ku70^(−/−) mice,cells from thymus, bone marrow and spleen were analyzed using monoclonalantibodies specific for lymphocyte surface markers and flow cytometry(19). Consistent with the immunohistological data there was a completeblock in B-cell development at the B220⁺CD43⁺ stage in the bone marrow,and there were no mature B cells in the spleen (FIG. 2B). In contrast,thymocytes developed through the CD4⁺CD8⁺ double-positive (DP) stage andmatured into CD4⁺CD8⁻ and CD4⁻CD8⁺ single-positive (SP), TCRβ positivecells (FIGS. 2B, C). In six four-week old Ku70^(−/−) mice analyzed, thepercentage of CD4-CD8⁻ double-negative thymocytes ranged from 11-62%,and the CD4⁺CD8⁺ DP cells varied from 35, 73%. CD4⁻CD8⁺ (1-11%) andCD4⁺CD8⁻ (1-3%) SP cells were also detected in the thymus. Furthermore,CD4⁺CD8⁻ or CD4⁻CD8⁺, single-positive T cells were found in the spleenin 67% of the mice studied (FIG. 2B), which expressed surface TCRβ (FIG.2C) and CD3. Thus, in contrast to the early arrest of both T- and B-cell development in Ku80^(−/−) mice (FIG. 2B), lack of Ku70 iscompatible with the maturation of T cells.

[0160] T-cell Receptor and Immunoglobulin Gene Rearrangement

[0161] To determine whether a null mutation in Ku70 affectsantigen-receptor gene recombination, DNA from bone marrow was amplifiedwith primers specific to immunoglobulin D-J_(H) and V-DJ_(H)rearrangements and DNA from thymus was amplified with primers thatdetected V-DJ_(β) and D_(δ)-J_(δ) rearrangements (20, 25-28). FIG. 3Ashows that Ku70^(−/−) B cells do undergo D-J_(H) recombination, at alevel which is similar to Ku80^(−/−) B cells, but is 2- to 3-fold lowerthan the level found in scid mice, and 10-50-fold lower than wild typelittermates. It is possible that some, but not all, of the decrease inD-J_(H) rearrangement is due to a lower fraction of B-lineage cells inthe mutant sample, since the wild type littermate mice have only ˜5-foldmore B220⁺ cells than the Ku70^(−/−) mice (see Table I). V-DJ_(H)rearrangements were not detected in either Ku70^(−/−), Ku80^(−/−), orscid bone marrow samples, possibly accounting for the absence of matureB cells in these mutant mice (FIG. 3A).

[0162] In contrast to the immunoglobulin heavy chain gene recombination,semiquantitative PCR analysis of thymocyte DNA for V-DJ_(β) jointsshowed normal levels of TCR_(β) rearrangements on a per cell basis (FIG.3B). Similarly, D_(δ)2 and J_(δ)1 coding joints were found in Ku70^(−/−)thymocytes at levels that resembled the wild type. To determine themolecular nature of the amplified coding joints, cloned V_(β)8-DJ_(β)2.6joints were sequenced. We found normal numbers of N, and P nucleotidesas well as normal levels of coding end deletions (FIG. 14). Thus, codingjoints in Ku70^(−/−) thymocytes differ from coding joints produced inxrs6 Ku80-deficient cells in that there were no large aberrant deletions(4, 18). We conclude that TCR V(D)J recombination in vivo does notrequire Ku70.

[0163] Absence of Ku70 Confers Radiation Hypersensitivity and Deficiencyin DNA DSB Repair

[0164] To assess radiation sensitivity in the absence of Ku70, cellsfrom the bone marrow were exposed to ionizing radiation, and wereassayed for colony formation (30, 32). FIG. 4A shows the survival curvesof the granulocyte/macrophage colony forming units (CFU-GM) fromKu70^(−/−), Ku80^(−/−) and wild type control mice. CFU-GM fromKu70-deficient mice were more sensitive to ionizing radiation than thosefrom Ku-proficient control mice (FIG. 4A). Similar hypersensitivity toradiation was seen for Ku80^(−/−) CFU-GM (FIG. 4A).

[0165] The rate and extent of rejoining of X-ray-induced DNA DSB inKu70^(−/−), Ku80^(−/−) and Ku70^(+/+) cells were measured usingasymmetric field inversion gel electrophoresis (AFIGE) (31). Fibroblastsderived from 13.5-day embryos were exposed to 40 Gy of X-rays andreturned to 37° C. for repair. At various times thereafter cells wereprepared for AFIGE to quantitate DNA DSB (FIG. 4B, upper panel). DNA DSBwere nearly completely rejoined in wild type cells within about 2 hafter radiation exposure. However, fibroblasts derived from Ku70^(−/−)mice showed a drastically reduced ability to rejoin DNA DSB. A similardeficiency in DNA DSB rejoining was also observed in fibroblasts derivedfrom Ku80^(−/−) embryos. Despite the large differences observed inrejoining of DNA DSB between wild type fibroblasts and fibroblastsderived from Ku70^(−/−) or Ku80^(−/−) mouse embryos, dose-responseexperiments showed that Ku70^(−/−), Ku80^(−/−) and wild type fibroblastswere equally susceptible to X-ray-induced damage (FIG. 4B, lower panel).Thus, Ku deficiency affects primarily the ability of cells to rejoinradiation-induced DNA DSB without significantly affecting the inductionof DNA damage.

[0166] Experimental Discussion

[0167] Absence of Ku70 results in radiation hypersensitivity,proportional dwarfism, as well as deficiencies in DNA DSB repair andV(D)J recombination. Thus, Ku70^(−/−) mice resemble Ku80^(−/−) mice inseveral respects but the two mutations differ in their effects on T andB cell development. Lack of Ku70 was compatible with TCR generearrangement and development of mature CD4⁺CD8⁻ and CD4⁻CD8⁺ T cells,whereas mature T cells were absent in Ku80^(−/−) mice. In contrast, Bcells failed to complete antigen receptor gene rearrangement and did notmature in either Ku70^(−/−) or Ku80^(−/−) mice.

[0168] What could account for the differences we find in TCR andimmunoglobulin gene rearrangements in the Ku70^(−/−) mice? Oneimplication of our findings is that there are alternativeKu70-independent rescue pathways that are compatible with completion ofV(D)J recombination in T cells. It is likely at the critical phase of Tcell maturation, other DNA repair activity may be stimulated (33, 34)and can functionally complement the Ku70 gene in T cell-specific V(D)Jrecombination. Since Ku80^(−/−) mice are deficient in both T and Blymphocyte development, it is plausible that these yet to be identifiedalternative DNA repair pathways include Ku80. The much reduced level ofKu80 protein in Ku70^(−/−) cells may in part account for thehypocellularity of Ku70^(−/−) thymii.

[0169] Although the role of Ku in V(D)J recombination is not molecularlydefined, Ku has been proposed to protect DNA ends from degradation (18,35), to activate DNA-PK (10, 11), and to dissociate the RAG/DNA complexto facilitate the joining reaction (20). These functions are notmutually exclusive, and they are all dependent on the interaction of Kuwith DNA. Thus, the finding that Ku70 is not required for TCR generearrangement is particularly unexpected, because the Ku70 subunit isbelieved to be the DNA-binding subunit of the Ku complex (36), andDNA-end binding activity was not detected in Ku70-deficient cells (FIG.1D).

[0170] In summary, our studies provide direct evidence supporting theinvolvement of Ku70 in the repair of DNA DSB and V(D)J recombination,and the presence of a Ku70-independent rescue pathway(s) in TCR V(D)Jrearrangement. The distinct phenotype of Ku70^(−/−) mice should makethem valuable tools for unraveling the mechanism(s) of DNA repair andrecombination.

[0171] References for the First Series of Experiments

[0172] 1. Li, Z., T. Otevrel, Y. Gao, H. -L. Cheng, B. Sneed, T.Stamato, G. Taccioli, and F. W. Alt. 1995. The XRCC4 gene encodes anovel protein involved in DNA double-strand break repair and V(D)Jrecombination. Cell 83: 1079-1089.

[0173] 2. Hendrickson, E. A., X. -Q. Qin, E. A. Bump, D. G. Schatz, M.Oettinger, and D. T. Weaver. 1991. A link between double-strandbreak-related repair and V(D)J recombination: The scid mutation. Proc.Natl. Acad. Sci. USA 88: 4061-4065.

[0174] 3. Pergola, F., M. Z. Zdzienicka, and M. R. Lieber. 1993. V(D)Jrecombination in mammalian cell mutants defective in DNA double-strandbreak repair. Mol. Cell. Biol. 13: 3464-3471.

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[0190] 17. Smider, V., W. K. Rathmell, M. R. Lieber, and G. Chu. 1994.Restoration of x-ray resistance and V(D)J recombination in mutant cellsby Ku cDNA. Science 266: 288-291.

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[0210] Second Series of Experiments

[0211] Recent investigations have linked the molecular mechanisms of twoprocesses, the repair of radiation-induced DNA double-strand breaks(DSB) and V(D)J recombination during T- and B-cell development. Themammalian DNA-dependent protein kinase DNA-PK has emerged as a keymolecule in these pathways. DNA-PK is a serine/threonine kinase thatconsists of a 465-kDa catalytic subunit (DNA-PKcs), and a DNA-targetingheterodimer consisting of a 70-kDa and an 86-kDa polypeptides (termedthe Ku70 and Ku80, respectively). When assembled on double-stranded DNAin vitro, the DNA-PK holoenzyme phosphorylates transcription factors andother proteins, including Sp1, Oct1, c-fos, c-jun, p53 and the 34-kDasubunit of replication protein A (Anderson, 1993; Pan, 1994). Geneticand biochemical studies strongly suggest a critical role for DNA-PK inDSB repair and V(D)J recombination (Jackson, 1995; Jeggo, 1995;Lees-Miller, 1996). Cell lines lacking either Ku80 or DNA-PKcs aredefective in both DSB repair and V(D)J recombination, and arehypersensitive to ionizing radiation (Blunt, 1995; Jackson, 1995; Jeggo,1995; Kirchgessner, 1995; Peterson, 1995; Rathmell, 1994; Smider, 1994;Taccioli, 1994). Genes encoding each of the subunits of DNA-PK have beenmapped to loci that complement the defect in x-ray-sensitive mutantcells (Jeggo, 1995; Thompson, 1995). The gene encoding DNA-PKcs maps tohuman chromosome 8q11, which is also identified as the locus of the SCIDgene (severe combined immune deficiency) (Blunt, 1995; Kirchgessner,1995; Sipley, 1995). Cells derived from SCID mice are hypersensitive tox-ray, defective in DSB repair and V(D)J recombination (Biedermann,1991), and lack DNA-PKcs expression (Blunt, 1995; Kirchgessner, 1995;Peterson, 1995). Consistent with these findings, a radiosensitive humanglioma cell line was found to be defective in DSB repair and devoid ofDNA-PKcs mRNA and proteins (Lees-Miller, 1995).

[0212] The Ku heterodimer was first discovered as an autoantigen inpatients with autoimmune disorders (Mimori, 1981). Genes encoding Ku70and Ku80 have been cloned and cytogenetically mapped to the humanchromosomes 22q13 and 2q33-35, (Cai, 1994). The groups of Dynan andJackson have provided evidence that Ku is the DNA-targeting subunit ofDNA-PK (Dvir, 1992; Gottlieb, 1993). Alone, neither DNA-PKcs nor Ku haskinase activity, and DNA-PK activity requires the assembly ofapproximately equimolar amounts of Ku70, Ku80 and DNA-PKcs ondouble-stranded DNA (Chan, 1996; Suwa, 1994).

[0213] Despite the rapid advances in our understanding of the geneticsof the DNA-PK subunits, the precise function of each of these proteinsin vivo, and their roles in DSB repair and V(D)J recombination remainunclear. Several models have been postulated (Jackson and Jeggo, 1995,Lees-Miller, 1996). After localization to a DSB, DNA-PK may signal viaphosphorylation to activate enzymes or other factors involved in therejoining of DNA ends. Alternatively, perhaps in addition to itsfunction in signaling, DNA-PK may structurally tether adjacent DNA endsin a conformation suitable for subsequent end rejoining (Jeggo, et al.,1995, Roth, et al., 1995). Although it remains to be proven, it is verylikely that the protein kinase activity of DNA-PK plays a critical rolein DNA repair and recombination (Jackson and Jeggo, 1995, Lees-Miller,1996). The in vivo function of Ku is also not well defined at themolecular level. Ku has been proposed to protect DNA ends fromdegradation (Liang and Jasin, 1996, Taccioli, et al., 1994), to activateDNA-PK (Dvir, et al., 1992, Gottlieb and Jackson, 1993) and todissociate the RAG/DNA complex to facilitate DNA joining reaction (Zhu,et al., 1996). These functions are not mutually exclusive, and they allappear to depend on the interaction of Ku with DNA molecules.

[0214] To facilitate studies on the function of the Ku subunits ofDNA-PK in vivo, we have recently carried out targeted disruption of Ku70and Ku80 genes in mice (Nussenzweig, et al., 1996, Ouyang, et al.,1997). In Ku80^(−/−) mice, the development of both T- and B-lymphocyteis arrested at early progenitor stages, and there is a profounddeficiency in V(D)J rearrangement (Nussenzweig, et al., 1996, Zhu, etal., 1996). Similar to Ku80^(−/−) phenotype, inactivation of Ku70 leadsto impaired B-lymphocyte development and deficient DSB repair (Ouyang,et al., 1997). However, in contrast to the Ku80^(−/−) phenotype, absenceof Ku70 does not abrogate T-cell receptor (TCR) gene recombination andthe development of mature T-cells (Gu, et al., 1997, Ouyang, et al.,1997). These studies indicate that Ku70 plays an essential role in DSBrepair, but is not essential for TCR V(D)J recombination, suggestingthat distinct and overlapping pathways may mediate DSB repair and V(D)Jrecombination. A related implication of these findings is that there maybe residual activity or alternate Ku70-independent pathways for V(D)Jrecombination during T-cell development. Hence, the processing of TCRV(D)J recombination in the Ku70^(−/−) mouse, which is defective in DSBrepair, may facilitate the generation of illegitimate recombinationevents (Cleary, 1991), potentially leading to tumor development.

[0215] In the present study, we examined the effect of the Ku70^(−/−)defect relative to malignant transformation and tumor development inmutant mice and derived cell lines. Fibroblasts derived from Ku70^(−/−)mice exhibit significantly higher frequencies of sister chromatidexchanges and spontaneous neoplastic transformation, relative to thewild type controls. Consistent with this cellular phenotype, themajority of Ku70^(−/−) mice developed spontaneous thymic anddisseminated T-cell lymphomas by 8 months of age. Lack of Ku70 proteinexpression was also found in more than 50% of tumor tissue specimensobtained from patients with T-cell lymphomas. Collectively, thesefindings strongly suggest the Ku70 locus as a candidate tumor suppressorgene for murine and human T-cell lymphomas.

[0216] We present evidence that inactivation of the Ku70 gene leads to apropensity for malignant transformation, both in vitro and in vivo.Ku70^(−/−) mouse fibroblasts displayed an increased rate of sisterchromatid exchange and a high frequency of spontaneous neoplastictransformation. Ku70^(−/−) mice, known to be defective in B- but notT-lymphocyte maturation, developed thymic and disseminated T-celllymphomas at a mean age of 6 months, with CD4⁺CD8⁺ tumor cellsexpressing wild type p53. A plausible link between Ku70 abnormality andhuman T-cell lymphomas was supported by the lack of Ku70 expression intumor specimens from four out of seven patients analyzed. These findingsdirectly demonstrate that Ku70-deficiency facilitates neoplastic growthand strongly suggest the Ku70 locus as a candidate tumor suppressorgene.

[0217] Experimental Results

[0218] Further Characterization of the Ku70^(−/−) Mouse

[0219] We have recently reported the generation of Ku70^(−/−) mice(Ouyang, et al., 1997). The Ku70 gene was inactivated by deleting 336-bpof exon 2, including the translational initiation codon of the mouseKu70 locus (FIG. 5A). Ku70^(−/−) heterozygotes exhibited noabnormalities and were used to generate a colony of Ku70^(−/−) mice,used for the current experiments.

[0220] PCR analysis using specific primers confirmed that part of exon 2was eliminated from the genome of Ku70^(−/−) offsprings (FIG. 5B), andWestern blot analysis with anti-Ku70 antibodies demonstrated the absenceof Ku70 protein in Ku70^(−/−) cells. Offsprings from Ku70^(+/−)intercrosses were of all three genotypes with approximately 25% beingKu70^(−/−) homozygotes, as expected from a Mendelian distribution.Ku70^(−/−) mice were fertile, but 40-60% smaller than their Ku70^(+/−)and Ku70^(+/+) littermates (FIG. 5C), a phenotype similar to Ku80^(−/−)mice (Nussenzweig, et al., 1996), but distinctly different from thatreported for SCID mice (Bosma, et al., 1983, Bosma and Carroll, 1991).The weight differences from the wild-type phenotype were present atbirth and maintained through adulthood (FIG. 5C).

[0221] Examination of tissues from Ku70^(−/−) mice revealedabnormalities in lymphatic organs and the gastrointestinal tract. Otherorgans, including brain, lung, liver, heart, kidney, testis and ovarieswere proportionally smaller but with no apparent structural orhistological abnormalities. Histological examination of thegastrointestinal tract showed mild to severe segmental aganglionosisaffecting small intestine and colon (discussed in a later section). TheKu70^(−/−) thymus was disproportionately smaller and contained 50- to100-fold fewer thymoctyes than Ku70^(+/+) littermates, but displayedrelatively normal appearing cortical-medullary junctions, as waspreviously reported (Ouyang, et al., 1997). The Ku70^(−/−) spleen wasalso 5- to 10-fold smaller with the splenic white pulp significantlyreduced. Immunohistochemical studies and multiparameter flow cytometricanalyses revealed that there was a complete block in B-cell developmentat early progenitor stages. In contrast, absence of Ku70 does not blockTCR gene rearrangement and the development of T-cells.

[0222] Ku70^(−/−) Mice Develop T-cell Lymphomas

[0223] As noted previously, the processing of V(D)J recombination andproliferation of T cell precursors in Ku70^(−/−) mouse, which has anintrinsic defect in DNA DSB repair, may enhance illegitimaterecombination and lead to tumor development. To test this hypothesis,the tumor susceptibility of Ku70^(−/−) mice was assessed. We randomlyassigned litters arising from heterozygous intercrosses (e.g.,Ku70^(+/+), Ku70^(+/−), Ku70^(−/−)) for our experiments and monitoredthe mice daily for tumor development and survival. As shown in FIG. 6,100% of Ku70^(+/+) (n=102) and Ku70^(+/−) (n=326) littermates remainedtumor-free and survived through the first 45 weeks of life. However, theactuarial survival of the Ku70^(−/−) mice at risk at 42 weeks was only22.4%, with a median survival of 28 weeks.

[0224] Autopsy examinations showed that, in the first 5-18 weeks oflife, 14.2% of Ku70^(−/−) mice died of severe forms of aHirschprung-like syndrome (see below). Subsequently, animals died ofthymic and disseminated lymphomas (FIG. 7). The youngest animal with adetectable tumor was 14 weeks old, and by 36 weeks of age, the greatmajority of the remaining Ku70^(−/−) mice died of lymphoma. Tumors of Blymphoid or non-lymphoid organs were not detected among the 45tumor-bearing animals examined. In contrast, for the same observationperiod, no tumors were detected in colonies of Ku80^(−/−) and SCID mice.Histologically, the primary tumors consisted of mononuclear, atypicalcells with cleaved nuclei, prominent nucleoli, and many mitotic figures.Immunohistochemical analyses revealed that the tumor cells were CD3⁺,confirming the diagnosis of T-cell lymphoma (FIGS. 7, D, E, and F). Inmost cases, these tumors involved other organs, such as the lung, heart,kidney, spleen and liver; a CD3⁺ phenotype was identified in all ofthese tumors.

[0225] Cell lines were readily established from five thymic tumors,designated T-96, T-49, T-248, T-311, and T-441. These lines had adoubling time of 16-18 hr. Flow cytometric analysis of three of thesetumor lines at early passage revealed a CD4⁺ CD8⁺ DP phenotype (FIG.7G), consistent with immature T cells of thymic origin. It is, thus,reasonable to postulate that some DP Ku70^(−/−) cells acquired mutationsthat enhanced their survival or the ability to proliferate relative tothat of short-lived wild type DP thymocytes.

[0226] Alterations of the p53 gene occur commonly in many differenttumor types, including lymphomas. To determine whether a similarphenotypic profile exists in the Ku70^(−/−) model, we analyzed the levelof p53 expression in the T-cell lymphomas developed in the Ku70^(−/−)mice. The normal cellular levels of p53 expression are very low andusually undetectable by immunohistochemical assays. In addition, thehalf-life of p53 protein is short, ranging from 5 to 20 minutes (Levine,1997). However, in tumors bearing dominant-negative mutations of p53,stabilization of the mutant protein leads to an accumulation of theprotein in the nuclei of tumor cells that can be readily detected byimmunohistochemical methods (Cordon-Cardo, et al., 1994). In allKu70^(−/−) T-cell lymphomas and their derived cell lines studied, wefound undetectable levels of p53 expression. Therefore, it can beconcluded that the wild type status of the p53 gene is maintained inthese murine T-cell lymphomas.

[0227] Ku70^(−/−) Fibroblasts also Undergo Malignant Transformation

[0228] Spontaneous neoplastic transformation occurs rarely in primarymouse fibroblasts. Consistent with this observation, primary mouse earfibroblasts (MEFs), derived from Ku70^(+/+) or Ku70^(+/−) and culturedup to passage 10, did not undergo spontaneous malignant transformation.In contrast, the formation of type III transformed foci was observed inKu70^(−/−) MEFs at a transformation frequency of 4.3×10⁻²/viable cell(FIG. 8, A and B). Co-transfection with HPV16 E6 and E7 into Ku70^(−/−)MEFs further increased the frequency of foci formation, whereastransformation was not observed in E6/E7 co-transfected Ku70^(+/+) orKu70^(+/−) fibroblasts.

[0229] Analysis of chromosomal aberrations in the various cell culturesgrown at 37° C. revealed that the Ku70^(−/−) cells contained 0.326sister chromatid exchanges (SCE) per chromosome (n=30 cells),representing a 2.2-fold increase over that of Ku70^(−/−) cells (0.147SCE per chromosome, n=34 cells) (p<0.05). Similarly, the E6/E7co-transfected Ku70^(−/−) cells contained a nearly 3-fold higherfrequency of SCE (0.262 SCE per chromosome, n=36 cells) than the E6/E7co-transfected Ku70^(+/+) or wild type Ku70^(+/+) cells (0.092 SCE perchromosome, n=23 cells) (p<0.05).

[0230] The foci derived from the primary and from the E6/E7co-transfected Ku70^(−/−) cultures were further tested for their abilityto grow under anchorage-independent conditions. FIG. 8C shows thatKu70^(−/−) cells derived from the transformed foci readily producedcolonies in soft agar, while no anchorage-independent growth was evidentfor the Ku70^(+/+) cells. Taken together, these results indicate thatKu70-deficiency leads to an increased propensity for malignanttransformation of primary mouse fibroblasts.

[0231] Extreme Radiation Sensitivity of Ku70^(−/−) Mice and Ku70^(−/−)Fibroblasts

[0232] We have shown previously that Ku70^(−/−) primary fibroblasts wereimpaired in the repair of radiation-induced DSB (Ouyang, et al., 1997).To demonstrate that this deficiency in DSB repair leads to thehypersensitivity of Ku70^(−/−) cells to radiation, monolayers ofKu70^(−/−) and Ku70^(+/+) primary ear fibroblasts (passage 7) wereexposed to graded doses of γ-irradiation (0-6 Gy), and survival wasdetermined by a colony formation assay. FIG. 9A clearly shows thatKu70^(−/−) cells were much more radiosensitive than the wild typecontrols, with a >100-fold difference in survival after 400 cGy ofγ-irradiation.

[0233] To assess the radiation-sensitive phenotype in vivo, adult (4months old) Ku70^(−/−) mice were given 400 cGy of γ-irradiation as werethe wild type controls (FIG. 9B). All wild type mice survived. However,all irradiated Ku70^(−/−) mice died within two weeks.

[0234] Gastrointestinal Abnormalities in Ku70^(−/−) Mice

[0235] In our experimental group of Ku70^(−/−) mice, we observed that14.2% died without evidence of lymphoma. Histological examination showedthat all these mice, as well as 60% of the lymphoma-bearing Ku70^(−/−)mice, showed unique gastrointestinal abnormalities. Mild to severesegmental aganglionosis was observed, affecting the small intestine andthe colon (FIG. 10). This phenotype was associated with the effacementof the typical morphology of the intestinal villi, dilatation ofintestinal lumens and denudation of the intestinal mucosa, causingfunctional obstruction and progressive distention of the intestine. Insome cases, we observed this alteration even in the esophagus andstomach. These changes were similar to those described in Hirschsprungdisease (Badner, et al., 1990). Death caused by the more severe form ofthis phenotype began around 5 weeks of age and peaked around 12 weeks,much earlier than the onset of lymphoma death at 14 weeks. Theseabnormalities were not observed in heterozygous and wild type mice up to8 months of age.

[0236] Ku70 Expression is Altered in Human T-cell Lymphomas

[0237] Because of the high incidence of T-cell lymphomas in Ku70^(−/−)mice, we evaluated the possibility that abnormal expression of Ku70 alsooccurs in human T-cell lymphomas. Seven patients with T-cell lymphomas,classified by a panel of antibodies to specific cell surface markers andmolecular probes, were analyzed. Immunohistochemical studies using apurified rabbit antiserum specific to Ku70, showed an intense nuclearstaining pattern of Ku70 protein in human normal lymphocytes of lymphnodes (FIG. 11A) and spleen samples. However, four of the seven T-celllymphomas analyzed showed undetectable Ku70 levels (FIG. 11C), while theremaining 3 cases displayed normal nuclear immunoreactivities (FIG.11B). In the four Ku70-negative cases, inflammatory cellular infiltratesin the periphery of the tumor were found to have a strong nuclearstaining, serving as internal positive controls.

[0238] We also analyzed the expression status of p53 in these seventumors. Of the four Ku70-negative T-cell lymphomas, three cases had anormal, undetectable level of p53 (FIG. 11F) and only one case displayeda mutated p53 phenotype. The remaining three T-cell lymphoma cases,which had a normal Ku70 expression (FIG. 11B), showed a mutated p53phenotype (FIG. 11E).

[0239] Experimental Discussion

[0240] The present study reveals a novel characteristic of theKu70^(−/−) phenotype, the propensity for malignant transformation, bothin vitro and in vivo. In vitro, this is expressed in terms of increasedrate of sister chromatid exchange, frequent spontaneous neoplastictransformation of primary fibroblasts and anchorage-independent growthof the transformed foci in soft agar. In vivo, Ku70^(−/−) micespontaneously develop thymic and disseminated T-cell lymphomas, althoughthe p53 phenotype in these tumors was normal. Concordant with thesedata, tumor specimens from human T-cell lymphomas also showed apathological lack of Ku70 protein. These findings directly demonstratethat inactivation of the Ku70 gene facilitates neoplastic growth, andstrongly suggest the Ku70 locus as a candidate tumor suppressor gene formurine and human T-cell lymphoma.

[0241] The specificity of the Ku70^(−/−) phenotype for the developmentof T-cell but not B-cell lymphoma is consistent with our recentobservation that the development of B-lymphocytes was absent inKu70^(−/−) mice (Ouyang, et al., 1997). In contrast to SCID andKu80^(−/−) mice, in which both T- and B-lymphocyte development isarrested at early progenitor stages (Bosma and Carroll, 1991, Carrolland Bosma, 1991, Carroll, et al., 1989, Lieber, et al., 1988,Nussenzweig, et al., 1996, Zhu, et al., 1996), the absence of Ku70blocks neither TCR gene rearrangement nor the development of mature Tcells (Gu, et al., 1997, Ouyang, et al., 1997). Nonetheless, the T-cellspecific differentiation was suboptimal in Ku70^(−/−) mice, with a 50-to 100-fold fewer thymocytes compared to the wild type littermates.These results suggest that there may be an alternate or residual, andKu70^(−/−) independent pathway for TCR V(D)J recombination andmaturation of T-cells, although it may be less efficient, or does notprovide all the necessary signals to fully effect the developmentaltransition. Another possible explanation for the lack of expansion ofKu70^(−/−) DP thymocytes may be associated with the intrinsic propensityof DP cells to undergo apoptosis (Smith, et al., 1989), which may befurther enhanced by the absence of Ku70. Consistent with this paradigm,we found that SV40-transfected Ku70^(−/−) cells were extremelysusceptible to radiation-induced apoptosis relative to wild typecontrols.

[0242] The mechanism for the induction of thymic lymphoma in Ku70^(−/−)mice is not clear at present. It is reasonable to hypothesize that athymocyte maturation defect and thymic malignancies are mechanisticallyrelated, and associated with abnormalities in DNA DSB repair, acharacteristic of the Ku70^(−/−) cells. Although residual DSB rejoiningmay be responsible for the apparent TCR V(D)J recombination, alternativeDNA repair pathways may exist in the absence of Ku70. Such pathways mayfunctionally complement the Ku70 gene and participate in TCR generearrangement. On the other hand, the rescue of TCR gene rearrangementand T-cell proliferation in a global DNA repair-deficient environmentmay result in unscheduled gene translocations, leading to thedevelopment of T-cell malignancies. Consistent with this model is ourcurrent observation on the increased frequency of neoplastictransformation in Ku70^(−/−) fibroblasts, suggesting that loss of Ku70may constitute one critical event in the multistep transformationprocesses.

[0243] The hypothesized link between deficient DSB repair, defectiveT-cell differentiation and tumor development in Ku70^(−/−) mice isconsistent with the experimental results obtained in irradiated SCIDmice (Danska, et al., 1994). While SCID cells were shown to be deficientin the repair of radiation-induced DSB and V(D)J recombination (Bosmaand Carroll, 1991, Carroll and Bosma, 1991, Carroll, et al., 1989,Lieber, et al., 1988), treatment of newborn SCID mice with a sublethalradiation dose of 100 cGy restored normal T-cell receptor TCRβrecombination, T-cell maturation and thymocyte proliferation, but notIgM rearrangement or B-cell development (Danska, et al., 1994). Relevantto this study is the observation that all of the irradiated SCID miceeventually developed T-cell tumors, but not tumors of B-lymphoid ornon-lymphoid origin. These data support the notion that the induction ofalternative pathways for DSB rejoining, apparently activated byradiation, can restore TCR V(D)J recombination, but because of theirdeficiency in DSB repair, these activities promote the malignanttransformation of T-cells. Therefore, the T-lineage specificity ofneoplastic transformation, either induced by low-dose irradiation (as inthe case of SCID mice) or occurring spontaneously (as in Ku70^(−/−)mice), may reflect an association between defective DNA DSB repair andTCR gene rearrangement.

[0244] Although Ku70^(−/−) cells of non-lymphoid lineage, such asprimary fibroblasts, can undergo spontaneous transformation in vitro, weobserved no spontaneous tumors other than T-cell lymphomas in theKu70^(−/−) mice. This may be due to the fact that nearly all animalsobserved up to the age of 8 months died of either T-cell lymphoma or aHirschsprung-like gastrointestinal syndrome. Mild to severe segmentalaganglionosis in the gastrointestinal tract was, in fact, detected inthe great majority of Ku70^(−/−) mice examined by autopsy. Thisunexpected phenotype was associated with the effacement of the typicalmorphology of the intestinal villi, dilatation of the intestinal lumensand denudation of the intestinal mucosa, disorders similar to thosedescribed in the Hirschsprung disease (HSCR). Human HSCR is a congenitaldisorder of the enteric nervous system characterized by the absence ofenteric ganglia (Badner, et al., 1990, Pingault, et al., 1997). Threegenes for HSCR have been identified, including the RET proto-oncogene(Angrist, et al., 1995, Attie, et al., 1995), the gene encoding theendothelin B receptor (EDNRB) (Amiel, et al., 1996), and the endothelin3 gene (EDN3) (Edery, et al., 1996, Hofstra, et al., 1996). In mice,spontaneous and in vitro-induced mutations affecting the RET, EDNRB, andEDN3 genes generate phenotypes similar to human HSCR. Another murinemodel of HSCR disease is the Dominant megacolon (Dom), a spontaneousmouse mutation in which the target gene has not yet been identified(Pavan, et al., 1995, Pingault, et al., 1997). Interestingly, the Dommutation has been mapped to the middle-terminal region of mousechromosome 15. Using known polymorphisms for conserved human/mousegenes, the homology between the Dom locus and human chromosome 22q12-q13has been established (Pingault, et al., 1997). Although the mouse Ku70locus is also mapped to chromosome 15 (Takiguchi, et al., 1996), it isunlikely that the Dom gene is disrupted in the Ku70^(−/−) mice, becauseof the fact that the homozygous Dom mutation results in a lethalphenotype. As the Dom gene sequences become available, it would be ofgreat interest to examine whether the expression of Dom gene, or that ofthe other HSCR genes, are affected by the absence of Ku70 protein.

[0245] The spontaneous development of T-cell tumors in the Ku70^(−/−)mice constitutes a major difference from the Ku80^(−/−) and SCIDphenotypes. It is, however, compatible with the thymic lymphoblasticlymphomas reported in Atm-deficient mice (Barlow, et al., 1996), thepredisposition to lymphoreticular malignancies in ataxia telangiectasiapatients (Boder, 1975, Sedgewick and Boder, 1991), and the developmentof thymic lymphoblastic lymphoma recently reported in DNA-PKcs null mice(Jhappan, et al., 1997). However, AT mutations are associated with othertumor types as well. Targeted disruption of one of the prototype tumorsuppressors, the p53 gene, also leads to the development of thymictumors in mice (Donehower, et al., 1992, Jacks, et al., 1994, Purdie, etal., 1994, Tsukada, et al., 1993). However, a dramatic susceptibility tothe development of other neoplasms, such as sarcomas, was also observedin p53^(−/−) mice (Donehower, et al., 1992, Jacks, et al., 1994).

[0246] The dominance of T-cell tumors in Ku70^(−/−) mice is unique,especially in view of their normal p53 phenotype. Our analysis of humantumor biopsies, however, suggests a possible association between Ku70and p53 in suppressing human T-cell lymphoma. We observed that 3 out of7 human T-cell lymphomas lacked Ku70 expression while showing a wildtype p53 phenotype, another three had the converse pattern with normalKu70 and abnormal p53 expression, and one tumor exhibited abnormalexpression of both genes. The lack of altered p53 expression in theKu70^(−/−) murine lymphomas, and the association of the human T-celllymphoma phenotypes with abnormalities in either Ku70 and/or p53 suggestthat they may serve in overlapping pathways of T-cell tumor suppression.

[0247] In summary, our studies show that inactivation of Ku70 results ina distinct phenotype, relative to Ku80^(−/−) and SCID mice, which aredeficient in the other component of the DNA-PK complex. Consistent withthe observation that the Ku70^(−/−) mouse is highly susceptible to thedevelopment of spontaneous thymic and disseminated T-cell lymphoma, mostof the human T-cell lymphomas examined also showed a lack of Ku70expression. Collectively, our studies directly demonstrate that thedisruption of Ku70 facilitates neoplastic growth and strongly suggestthat the Ku70 locus as a candidate tumor suppressor gene for murine andhuman T-cell lymphoma. Although the Ku70^(−/−) rodent model did notexhibit other types of tumor, the high frequency of sister chromatidexchanges in Ku70^(−/−) fibroblasts and their high susceptibility tospontaneous neoplastic transformation raises the possibility that otherhuman tumors may also be affected by the function of the Ku70 locus.Further experiments will be required to assess this possibility.

[0248] Experimental Procedures

[0249] Target Disruption of Ku70 and Generation of Ku70^(−/−) Mice

[0250] Mouse genomic Ku70 gene was isolated from a sCos-I cosmid libraryconstructed from a mouse strain 129 embryonic stem cell line (Takiguchi,et al., 1996). The replacement vector was constructed using a 1.5 kb5′-fragment which contains the promoter locus with four GC-box and exon1, and a 8 kb EcoRV-EcoRI fragment extending from intron 2 to intron 5as indicated in FIG. 1a. Homologous replacement results in a deletion of336-bp exon 2 including the translational initiation codon.

[0251] The targeting vector was linearized with Not 1 and transfectedinto CJ7 embryonic stem (ES) cells by electroporation using a Bio-RadGene Pulser. Three hundred ES cell clones were screened, and 5 clonescarrying the mutation in Ku70 were identified by Southern blotting.Positive ES clones were injected separately into C57BL/6 blastocysts togenerate chimeric mice. One clone was successfully transmitted throughthe germline after chimeras were crossed with C57 BL/6 females.Homozygous Ku70^(−/−) mice were generated by intercrossing Ku70^(+/−)heterozygotes.

[0252] The genotypes of the mice were first determined by tail PCRanalysis which distinguishes endogenous from the targeted Ku70 allele,and subsequently confirmed by Southern blot analysis. The PCR reactioncontained 1 μg genomic DNA; 0.6 μM (each) of primers HO-2:GGGCCAGCTCATTCCTCCACTCATG, HO- 3: CCTACAGTGTACCCGGACCTATGCC and HO-4:CGGAACAGGACTGGTGGTTGAGCC; 0.2 mM (each) dNTP; 1.5 mM MgCl₂ and 2.5 U ofTaq polymerase. Cycling conditions were 94° C. for 1 min, 64° C. for 1min, 72° C. for 1 min (30 cycles), followed by an extension at 72° C.for 10 min. Primers HO-2 and HO-4 give a product of the targeted allelethat is ˜380 bp; primers HO-3 and HO-4 yield a wild type product of 407bp.

[0253] Cell Cultures and Determination of Radiosensitivity

[0254] Monolayers of cells (1-2×10⁵ cells) were seeded in 60 mm petridishes and cultured at 37° C. for 3 days at which time they were nearconfluence (1-2×10⁶ cells per dish). The culture medium was then changeddaily, and the cells were at a density-inhibited plateau phase by day 6.The pulse-labeling index, as determined by incubation for 30 min with 10μCi/ml of ³H-thymidine and autoradiographic analysis, was <1% indicatinga paucity of cycling cells. Experiments were performed on day 6 or 7.

[0255] Survival curves were obtained by measuring the colony-formingability of irradiated cells as described previously (Nagasawa, et al.,1991). A colony containing more than 50 cells was scored as a survivor.Cell survival was always normalized to the cloning efficiency ofuntreated controls. All experiments were performed at least three timesand yielded consistent results.

[0256] Spontaneous Transformation of Ku70-Deficient Cells

[0257] To study the spontaneous transformation of Ku70-deficientfibroblasts, the well established protocols of Little were used (Little,1979). Cells were seeded into 6 replicate 100-mm plastic Falcon petridishes, at densities designed to yield approximately 4000 to 7000 viable(colony forming) cells per dish. After a 3- to 4-week incubation at 37°C., with twice weekly renewal of the nutrient medium, the cultures werefixed with 95% ethanol and stained with 0.1% crystal violet. Transformedfoci (Type III) appeared as dense piled-up colonies of cells overlyingthe normal monolayer. Cells from these foci were isolated, expanded andfurther tested for their ability to grow in soft agar in ananchorage-independent manner.

[0258] In parallel with the above, three 100 mm dishes were seeded froma 1:50 dilution of the same cell suspension (80 to 140 viable cells) ineach group in order to determine the actual colony forming efficiency.After a 10- to 12-day incubation at 37° C., the samples were fixed andstained, the number of viable colonies counted, and the cloningefficiency determined, which was then used to calculate the number ofviable cells seeded in the transformation dishes. The transformationfrequency was determined by dividing the total number of transformedfoci scored in a treatment group by the total number of viable cellsseeded, and it was therefore expressed as transformants per viable cell.

[0259] For colony formation in soft agar, a modified MacPherson method(MacPherson, 1973) was used (Nagasawa, et al., 1987). Plastic petridishes (60 mm) were coated with a layer of 5 ml of 0.5% agarose inmedium supplemented with 20% heat-inactivated fetal bovine serum. Twomilliliters of the cell suspension were mixed with 4 ml of the 0.5%agarose solution; 1.5 ml of the resulting cell suspension were platedinto the agarose-coated dishes. Subsequently, the cultures were fed oncea week by adding 1 ml of complete medium (without agarose). The size ofthe colonies was monitored at 2 days, 1, 2, and 3 weeks after seeding bytaking photomicrographs of the cultures on an inverted microscope.

[0260] Analysis of Sister Chromatid Exchange

[0261] For analysis of sister chromatid exchange (SCE), the protocolsused by Nagasawa et al (Nagasawa, et al., 1991) were followed. Briefly,cells were subcultured from density-inhibited cultures into threereplicate T-25 tissue culture flasks in fresh complete medium containing10⁻⁵ M bromodeoxyuridine (BrdUrd) for two rounds of cell replication.For three successive 4-h intervals beginning 15 h after subculturing,colcemid (0.2 g/ml) was added to one of the flasks for a 4-h intervalprior to fixation. Therefore, harvesting was carried out over a totalperiod of 12 h. Chromosomes were prepared for the analysis of SCE by theair-dry method, as previously described (Nagasawa and Little, 1979,Nagasawa, et al., 1991). The differential staining of sister chromatidswas carried out by the fluorescence plus Giemsa technique (Nagasawa, etal., 1991, Perry and Wolff, 1974). SCE was analyzed at peak mitoticindices after completion of the first or second mitosis.

[0262] Immunohistochemistry

[0263] Normal and tumor tissue samples from wild type and/or Ku70^(−/−)mice were fixed in either 10 buffered formalin and embedded in paraffin,or embedded in OCT compound (Miles Laboratories) and frozen in liquidnitrogen at −70° C. Seven human T-cell lymphomas were analyzed, as wellas human normal tissue samples of lymph node, spleen, and colon.Sections (5 μm) were stained with hematoxylin and eosin, andrepresentative samples were selected for immunohistochemical analysis.Immunophenotyping was performed using an avidin-biotin immunoperoxidasetechnique (Cordon-Cardo and Richon, 1994, Serrano, et al., 1996).Primary antibodies included anti-mouse CD45 (purified rat monoclonalantibody, 1:500, PharMingen), anti-mouse CD3 (purified rabbit serum,1:1000, Dako), anti-mouse B220 (purified rat monoclonal antibody,1:1000, PharMingen), and anti-mouse CD19 (purified rat monoclonalantibody, 1:1000, PharMingen), and were incubated overnight at 4° C. Wealso used a purified rabbit antiserum to the Ku70 nuclear protein (1:500dilution), and a purified sheep antiserum that recognizes murine p53(Calbiochem, 1:1000). Samples were subsequently incubated withbiotinylated secondary antibodies (Vector Laboratories) for 30 min (goatanti-rabbit, 1:500; rabbit anti-rat, 1:100; rabbit anti-sheep, 1:400),and then with avidin-biotin peroxidase complexes (1:25 dilution, VectorLaboratories) for 30 min. Diaminobenzadine was used as the chromogen andhematoxylin as the counter stain. Wild type lymphoid organs includingthymus, spleen and lymph nodes from different mice were used fortitration of the antibodies and positive controls. For negativecontrols, primary antibodies were substituted with class-matched butunrelated antibodies at the same final working dilutions (Ouyang, etal., 1997).

[0264] Flow Cytometry Analysis of the Spontaneous Tumors

[0265] Cell lines were established from each primary tumor as follows.Samples of the tumors were dispersed into cell suspension and plated atvarious densities in RPMI supplemented with 10% heat-inactivated fetalbovine serum and antibiotics. The cell cultures were split 1:2 and 1:4until they become established.

[0266] For flow cytometry analysis, tumor cells of early passages werestained with combinations of antibodies specific for various T- andB-lymphocyte surface markers, such as PE-labeled anti-mouse CD4, andFITC-labeled anti-mouse CD8, and analyzed on a Becton Dickinson FAC scanwith Cell Quest software (Ouyang, et al., 1997).

[0267] References for the Second Series of Experiments

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[0323] 56. Smider, V., W. K. Rathmell, M. R. Lieber and G. Chu (1994)Restoration of x-ray resistance and V(D)J recombination in mutant cellsby Ku cDNA. Science, 266, 288-291.

[0324] 57. Smith, C. A., G. T. Williams, R. Kingston, E. J. Jenkinsonand J. J. T. Owen (1989) Antibodies to CD3/T-cell receptor complexinduce death by apoptosis in immature T cells in thymic cultures. Nature(London), 337, 181-184.

[0325] 58. Suwa, A., M. Hirakata, Y. Takeda, S. A. Jesch, T. Mimori andJ. A. Hardin (1994) DNA-dependent protein kinase (Ku protein-p350complex) assembles on double-stranded DNA. Proc. Natl. Acad. Sci. USA,91, 6904-6908.

[0326] 59. Taccioli, G. E., T. M. Gottlieb, T. Blunt, A. Priestly, J.Demengeot, R. Mizuta, A. R. Lehmann, F. A. Alt, S. P. Jackson and P. A.Jeggo (1994) Ku80: product of the XRCC5 gene and its role in DNA repairand V(D)J recombination. Science, 265, 1442-1445.

[0327] 60. Takiguchi, Y., A. Kurimasa, F. Chen, P. E. Pardington, T.Kuriyama, R. T. Okinaka, R. Moyzis and D. J. Chen (1996) Genomicstructure and chromosomal assignment of the mouse Ku70 gene. Genomics,35, 129-135.

[0328] 61. Thompson, L. H. and P. A. Jeggo (1995) Nomenclature of humangenes involved in ionizing radiation sensitivity. Mutat. Res., 337,131-133.

[0329] 62. Tsukada, T., Y. Tomooka, S. Takai, Y. Ueda, S. Nishikawa, T.Yagi, T. Tokunaga, N. Takeda, Y. Suda, S. Abe, I. Matsuo, Y. Ikawa andS. Aizawa (1993) Enhanced proliferative potential in culture of cellsfrom p53-deficient mice. Oncogene, 8, 3313-3322.

[0330] 63. Zhu, C., M. A. Bogue, D. -S. Lim, P. Hasty and D. B. Roth(1996) Ku86-deficient mice exhibit severe combined immunodeficiency anddefective processing of V(D)J recombination intermediates. Cell, 86,379-389.

[0331] Third Series of Experiments

[0332] Ku-Deficient Cells are Sensitive to γ-Rays and ChemotherapeuticAgents

[0333] Survival experiments using cells derived from either Ku70 or Ku80knock-out mice have shown that these cells are very sensitive toγ-radiation and several chemotherapeutic agents, specifically thoseagents that induce DNA strand breaks, such as: bleomycin, etoposide, andadriamycin (FIG. 12).

[0334] HSP70 Promoter Analysis

[0335] Experiments were performed to test the transcriptional activityof the mouse hsp70 promoter. For these experiments, first, the plasmidN3Luc, a reporter gene construct which contains the mouse hsp70 promoterupstream of the firefly luciferase gene was used for our studies. Cellswere transiently transfected with this mouse hsp70 promoter-drivenluciferase reporter gene construct. Comparison of the luciferaseactivity before and 8 hours after heat shocking the cells demonstratedthat a) this promoter showed little “leakiness” (i.e. low transcriptionunder normal conditions) and b) a high heat-inducible activity. Thetranscriptional activity after a 15 minute 45° C. heat shock was atleast 30 fold increased relative to control levels. Other investigatorshave reported even higher induction levels (>100 fold) for this promoter(Nguyen et al., J. Biol. Chem. 264: 10487 (1989)).

[0336] Mutant of the hsp70 promoter were then generated, including5′-deletion, linkerscanner mutations and point mutations, fused to thefirefly luciferase reporter gene (the mutant N3Luc construct isdesignated ΔN3Luc), and examined the heat-induced reporter geneexpression. Our results showed that specific deletion (e.g., either at5′ or in the central region of hsp70 promoter) increased the heatinduction of transcriptional activity (as measured by firefly luciferasereporter gene activity) by an additional several fold when compared tothe heat inducibility of the intact, not mutated promoter. Further dataindicate that in cells deficient in Ku70 or Ku80 the heat induction ofhsp7o promoter activity is further enhanced.

[0337] Stable HeLa cells, containing human Ku70 cDNA or human Ku80 cDNA,in the antisense orientation, under the regulation of the Tet-Off™expression system (Clonetech), were established. Upon induction of theexpression system these cells should produce antisense Ku70 or Ku80 RNA,respectively. Experiments were performed showing (FIG. 13) thatexpression of either Ku70 or Ku80 antisense RNA increased the cytotoxiceffect adriamycin by 3-5 fold at 1 μg/ml and that expression of Ku70antisense RNA increased the cytotoxic effect of γ-radiationapproximately 5 fold (at 6 Gy).

What is claimed is:
 1. A method of diagnosing a predisposition to cancerin a subject comprising: a. obtaining a nucleic acid sample from thesubject; and b. determining whether one or more of the subject's Ku70alleles or regulatory regions to those alleles are deleted or differentfrom the wild type so as to reduce or eliminate the subject's expressionof polypeptide having tumor suppressor activity.
 2. The method of claim1, wherein the cancer is T-cell lymphoma.
 3. The method of claim 1,wherein the cancer is B-cell lymphoma.
 4. The method of claim 1, whereinthe cancer is neuroblastoma.
 5. The method of claim 1, wherein theregulatory region is a promoter.
 6. The method of claim 1, wherein thedetermining of step b comprises generating a polypeptide encoded by oneor more of the subject's Ku70 alleles and comparing the resultingpolypeptide to a wild type Ku70 polypeptide.
 7. The method of claim 1,wherein the nucleic acid sample is obtained from the subject's blood. 8.A method of diagnosing a predisposition to cancer in a subjectcomprising: determining the level of Ku70 expression in the subject. 9.The method of claim 8, wherein the cancer is T-cell lymphoma.
 10. Themethod of claim 8, wherein the cancer is B-cell lymphoma.
 11. The methodof claim 8, wherein the cancer is neuroblastoma.
 12. The method of claim8, wherein the level of Ku70 expression is determined based upon thelevel of Ku70 mRNA in the subject.
 13. The method of claim 8, whereinthe level of Ku70 expression is determined based upon the level of Ku70polypeptide in the subject.
 14. The method of claim 8, wherein zero orreduced Ku70 expression indicates a predisposition to cancer.
 15. Amethod of diagnosing a predisposition to cancer in a subject comprising:determining the subcellular localization of Ku70 in the subject, whereinan abnormal subcellular localization of Ku70 indicates a predispositionto cancer.
 16. The method of claim 15, wherein the abnormal subcellularlocalization of Ku70 comprises increased cytosolic localization of Ku70.17. The method of claim 15, wherein the abnormal subcellularlocalization of Ku70 comprises decreased nuclear localization of Ku70.18. A method of assessing the severity of cancer in a subjectcomprising: a. obtaining a nucleic acid sample from the subject; and b.determining whether one or more of the subject's Ku70 alleles orregulatory regions to those alleles are deleted or different from thewild type so as to reduce or eliminate the subject's expression ofpolypeptide having tumor suppressor activity.
 19. The method of claim18, wherein the cancer is T-cell lymphoma.
 20. The method of claim 18,wherein the cancer is B-cell lymphoma.
 21. The method of claim 18,wherein the cancer is neuroblastoma.
 22. A method of assessing theseverity of cancer in a subject comprising: determining the subcellularlocalization of Ku70 in the subject, wherein an abnormal subcellularlocalization of Ku70 indicates a predisposition to cancer.
 23. Themethod of claim 22, wherein the abnormal subcellular localization ofKu70 comprises increased cytosolic localization of Ku70.
 24. The methodof claim 22, wherein the abnormal subcellular localization of Ku70comprises decreased nuclear localization of Ku70.
 25. A method ofinhibiting the growth of cancer cells, comprising introducing into acell a Ku70 gene under conditions permitting expression of the gene. 26.The method of claim 25, wherein the cancer is T-cell lymphoma.
 27. Themethod of claim 25, wherein the cancer is B-cell lymphoma.
 28. Themethod of claim 25, wherein the cancer is neuroblastoma.
 29. The methodof claim 25, wherein the cell prior to the introduction of the Ku70 genewas characterized as having a mutation at one or more Ku70 alleles orregulatory regions thereto.
 30. The method of claim 29, wherein themutation is a frameshift mutation.
 31. The method of claim 29, whereinthe mutation is a point mutation.
 32. The method of claim 25, whereinthe cell prior to the introduction of the Ku70 gene was characterized ashaving reduced expression of Ku70.
 33. The method of claim 25, whereinthe Ku70 gene is incorporated into an expression vector prior tointroduction into the cell.
 34. A method of inhibiting the growth ofcancer cells, comprising introducing Ku70 into a cell.
 35. The method ofclaim 34, wherein the cancer is T-cell lymphoma.
 36. The method of claim34, wherein the cancer is B-cell lymphoma.
 37. The method of claim 34,wherein the cancer is neuroblastoma.
 38. A transgenic cell, wherein theexpression of the Ku70 allele has been altered to increase thesusceptibility of the cell to DNA damage.
 39. A transgenic cell, whereinthe expression of the Ku70 allele has been altered to increase thesusceptibility of the cell to cancerous growth.
 40. A transgenicorganism, comprising an organism whose germ line cells has been alteredat the Ku70 allele to produce an organism whose offspring have anincreased likelihood of developing tumors.
 41. A transgenic organism,comprising an organism whose germ line cells has been altered at theKu70 allele to produce an organism whose offspring have an increasedlikelihood of having increased susceptibility to DNA damage.
 42. Thetransgenic organism of claim 40, wherein the somatic cells have beenaltered to reduce or eliminate expression of Ku70.
 43. The transgenicorganism of claim 41, wherein the somatic cells have been alterred toreduce or eliminate expression of Ku70.
 44. The transgenic organism ofclaim 40, wherein the organism is a mouse.
 45. The transgenic organismof claim 41, wherein the organism is a mouse.
 46. A method of screeninga compound for carcinogenic activity, comprising: a. contacting a cellhaving reduced expression of Ku70 with the compound; and b. determiningwhether the compound results in a malignant transformation phenotype.47. The method of claim 46, wherein the cell is a fibroblast.
 48. Themethod of claim 46, wherein the malignant transformation phenotypecomprises anchorage independent growth.
 49. A method of screening acompound for DNA damaging activity, comprising: a. contacting a cellhaving reduced expression of Ku70 with the compound; and b. determiningwhether the compound results in DNA damage.
 50. The method of claim 49,wherein the DNA damage is determined by measuring a reduction in cellsurvival.
 51. The method of claim 49, wherein the DNA damage comprisesone or more double strand breaks.
 52. A method of screening a compoundfor ability to restore Ku70 activity to cells having Ku70 defectsymptoms resulting from reduced Ku70 activity, comprising: a. contactinga cell having reduced expression of Ku70 with the compound; and b.determining whether the compound restores, in whole or in part, a normalKu70 phenotype.